1 Department of Microbiology, University of Pennsylvania, 3610 Hamilton Walk,
211 Johnson Pavilion, Philadelphia PA 19104, USA
2 MRC-Laboratory for Molecular Cell Biology and Department of Biochemistry and
Molecular Biology, University College London, Gower Street, London WC1E 6BT,
UK
3 Department of Cell Biology, Building NC1, Lerner Research Institute, Cleveland
Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio 44195, USA
* Author for correspondence (e-mail: guvakova{at}mail.med.upenn.edu)
Accepted 19 August 2002
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Summary |
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Key words: IGF-IR signaling, -actinin, Actin cytoskeleton, Breast cancer cell migration
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Introduction |
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Some mechanistic aspects and common sequential changes that precede cell
separation have been determined using animal and cellular models (reviewed in
Hay, 1995;
Thiery and Chopin, 1999
;
Savagner, 2001
). For example,
epithelial colonies can be induced to separate by treatment with physiological
peptides, including hepatocyte growth factor/scatter factor (HGF/SF),
neuregulin, members of the fibroblast growth factor (FGF), epidermal growth
factor (EGF) and transforming growth factor (TGF) families
(Stoker and Perryman, 1985
;
Savagner et al., 1997
;
Chausovsky et al., 1998
;
Müller et al., 1999
).
Peptides with cell scattering function induce a disruption of intercellular
junctions, a reorganization of the actin cytoskeleton and an increase in local
cell motility. These effects are mediated by receptor tyrosine kinases
(Sachs et al., 1996
), of which
the most extensively studied is Met, the receptor for HGF/SF (reviewed in
Furge et al., 2000
).
Activation of Met has a strong effect on scattering and invasiveness of the
rodent mammary epithelial cells and various human carcinoma cell lines, and
similar disorganization is believed to lead to metastasis (Wiedner et al.,
1990; Liang et al., 1996
;
Firon et al., 2000
). It is
less clear, however, whether HGF/SF and Met control motile behavior of human
breast tumor cells. No scattering response to HGF/SF has been seen in human
breast carcinoma cell lines in vitro
(Stoker et al., 1987
); in
breast tissue biopsies, the intensity of Met staining was lower in tumor
tissue than in normal cells adjacent to mammary ducts
(Tsarfaty et al., 1992
).
Another member of the receptor tyrosine kinase family is the receptor for
IGF-I, a protein of particular interest for the biology of the human mammary
gland. There is a general requirement for IGF-I in terminal end bud formation
and ductal morphogenesis in the developing gland
(Kleinberg et al., 2000). In
humans, the IGF-IR is expressed by both normal and tumor mammary cells; yet
the level of the receptor expression and its tyrosine kinase activity is
higher in malignant compared with benign and normal tissues
(Happerfield et al., 1997
;
Resnik et al., 1998
). The
signaling mechanisms evoked by IGF-IR kinase are well documented (reviewed by
LeRoith, 2000
). Ligand-induced
activation of the IGF-IR causes the sequential phosphorylation of three
conservative tyrosines within the catalytic domain, followed by
phosphorylation of the cytoplasmic domain of the receptor, recruitment of
adapter molecules and initiation of signaling cascades. The two
best-characterized pathways triggered by the activation of this receptor are
the PI3-kinase signaling via the insulin receptor substrates (IRSs) and the
MAP kinase pathway via adaptor molecules Shc. Although IGF-I may have a
pleiotropic effect on cell proliferation, differentiation and migration
(Leventhal and Feldman, 1997
;
Lee et al., 1998
;
Chernicky et al., 2000
), the
range of IGF-IR activities is critically dependent on the cell context
(Baserga, 1999
). There is good
evidence that in cultured mammary epithelial cells the IGF-IR stimulates not
only proliferative but also motile cell responses
(Kohn et al., 1990
;
Doerr and Jones, 1996
;
Mira et al., 1999
). We have
recently reported that IGF-I treatment of breast cancer cells overexpressing
IGF-IR results in a disruption of the epithelial sheet, implying a role for
this receptor in concomitant reorganization of the actin cytoskeleton and
cellular junctions (Guvakova and Surmacz,
1999
).
The most prominent epithelial structures relying on the actin cytoskeleton
are adherence junctions. The core of these junctions is composed of
transmembrane E-cadherins and cytoplasmic ,ß- and
-catenins
(reviewed by Gumbiner, 2000
).
-catenin directly or via actin-binding proteins
-actinin and
vinculin couples cadherin-catenin complexes to microfilaments
(Knudsen et al., 1995
;
Nieset et al., 1997
;
Vasioukhin et al., 2000
). Such
a linkage is essential for the stabilization of intercellular adhesion and the
maintenance of mammary epithelial tissue
(Tsukatani et al., 1997
). On
the basis of the correlation between upregulation of the IGF-IR in breast
tumors and the effects of the overexpressed IGF-IR on the actin cytoskeleton,
we hypothesize that high levels of IGF-IR activity can induce destabilization
of cell-cell contacts. We report a novel signaling effect of the IGF-IR kinase
in coupling
-actinin and actin-containing microspikes by a PI
3-kinase-dependent mechanism at an early stage of epithelial cell
separation.
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Materials and Methods |
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Molecular cloning and cell transfection procedures
MCF-7/IGF-IR/DK kinase-deficient cells were generated using a IGF-IR ß
subunit with mutations in the activation loop of the catalytic domain (three
tyrosine residues, Y1131, Y1135, and Y1136 were substituted by
phenylalanines). This DNA was a gift from D. Leroith, NIH
(Kato et al., 1994). The
mutant IGF-IR cDNA was subcloned from the pBluscript II KS+ vector into the
EcoRI and XbaI sites of the pcDNA3 expression vector
(Invitrogen Corp., San Diego, CA) under the control of the CMV promoter. MCF-7
cells expressing the mutant receptor were derived by
calcium-phosphate-precipitation-mediated transfection with a plasmid encoding
the mutant hIGF-IR followed by selection in 2.5 mg/ml G418. Surface expression
of the IGF-IR was confirmed by fluorescence-activated cell sorting (FACS)
using a monoclonal antibody against the
subunit of the IGF-IR and a
secondary FITC-conjugated goat anti-mouse antibody (Vector). Stable clones
expressing approximately 1.0x106 IGF-IRs per cell were
isolated.
For functional analysis of -actinin, new DNA constructs encoding the
truncated
-actinins with enhanced green fluorescent protein (EGFP)
tagged to the C-terminus were generated. A full-length chicken sarcomeric
-actinin (GenBankTM accession number X59247) was obtained from J.
Sanger (University of Pennsylvania)
(Dabiri et al., 1997
). A 230
amino-acid N-terminal truncation, including residues 1-24 in the potential
site for regulation of high-affinity binding of
-actinin to actin
(Xu et al., 1998
) and residues
108-189 in the actin-binding site determined by mutagenesis
(Hemmings at al., 1992
), was
produced by excision of a unique 5' Eco47III-EcoRV
restriction enzyme fragment, followed by blunt-end religation of the truncated
plasmid DNA. In this mutant
-actinin RNA, the authentic methionine
start codon is removed, and the translation is initiated at the AUG codon for
methionine 231. The deletion of the C-terminal tail of
-actinin was
generated by excision of a SacI fragment from the plasmid encoding
full-length
-actinin, followed by religation of the fragment into the
SacI site of pEGFPN1 vector. The authentic initiating codon is
retained in this mutant. The removed region encodes 168 amino acids, including
a large part of a vinculin-binding site [residues 713-749
(McGregor et al., 1994
)] and
both EF-hand Ca2+-binding motifs
(Baron et al., 1987
). The
proper deletions were confirmed by restriction mapping of the N- and C-
terminal regions of
-actinin cDNA. The position of the truncated
-actinins in frame with EGFP was verified using the Vector NTI version
3.0 program (InforMax, Inc.).
In transient transfection experiments, 5x104
MCF-7/IGF-IR/WT or MCF-7/IGF-IR/DK cells were plated into a 35 mm
glass-bottomed culture dish (MatTek Corporation, Ashland, MA). The next day,
the cells were transfected with plasmids encoding EGFP--actinin
constructs or the pEGFP-N1 control vector (Clontech) using Lipofectamine Plus
Reagent (Gibco, BRL). After 3 hours, the cells were washed in D-MEM/F-12 and
incubated overnight in the conditioned medium of MCF-7/IGF-IR/WT cells to
improve the survival of the transfectants. The living cells were used in
experiments 48 hours after transfection, when approximately 40% of cells
produced fluorescently labeled protein.
Western blotting and immunoprecipitation
Cells were lysed in buffer containing 50 mM Hepes pH 7.5, 150 mM NaCl, 1.5
mM MgCl, 1 mM EGTA, 10% glycerol, 20 µg/ml aprotinin, 1 mM
phenylmethylsulfonyl fluoride, 2 mM Na orthovanadate and either 1% Triton
X-100 or 1% SDS. For western blots, 20 µg of total protein was resolved by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Hybond
ECL nitrocellulose (Amersham Pharmacia Biotech). In a series of experiments,
the blots were probed with antibodies against IGF-IR (rabbit polyclonal
anti-IGF-IR ß subunit, Santa Cruz, SC); IRS-1 (rabbit polyclonal
anti-IRS-1, Upstate Biotechnology Inc., UBI, Lake Placid, NY); -actinin
(mouse IgG clone AT6/172, UBI and goat polyclonal anti-
-actinin
antobodies, SC);
-catenin (rabbit polyclonal antibodies, Sigma); GFP
(mouse monoclonal antibody, Clontech Laboratories, Inc.); phospho-Akt (rabbit
anti-phospho-Akt (Ser 473), rabbit anti-phospho-Akt (Thr 308), New England
Biolab, NEB); Akt (rabbit anti-Akt, NEB); phospho MAPK (mouse monoclonal E10,
anti-phospho-p44-42 MAPK (Thr 202/Tyr 204), NEB); ERK1/ERK2 (rabbit polyclonal
k23 anti-ERK1, SC), and phospho-tyrosine-containing proteins (mouse monoclonal
PY-20 anti-phosphotyrosine, SC). For immunoprecipitation of
-actinin,
500 µg of total protein from 1% SDS cellular extracts was incubated with 4
µg of anti-
-actinin monoclonal IgG1, clone AT6/172 (UBI)
and 50 µl anti-mouse IgG-agarose beads (Sigma) in HNTG buffer (20 mM Hepes
pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 10% glycerol) overnight at 4°C.
The immunoprecipitated proteins were resolved by SDS-PAGE and blotted with
anti-
-actinin and PY 20 antibodies. To visualize primary antibody-bound
protein, the secondary antibodies conjugated to horseradish peroxidase
(1:10,000 dilution; Calbiochem) and the ECL detection solutions (Amersham
Pharmacia, UK) were applied. The chemiluminescent intensity of the bands was
digitized using the Image Analyser LAS-1000 plus system and the Image Reader
LAS-1000 Lite version 1.0 software (Fuji). When several proteins were to be
detected on the same membrane, nitrocellulose was incubated in stripping
buffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl pH 6.7) for 30
minutes at 50°C, then washed three times in washing buffer (100 mM Tris pH
7.5, 1M NaCl, 1% Tween-20), blocked in 5% BSA in PBS and re-probed. The
relative activity of the protein kinase B (PKB/Akt) and mitogen-activated
protein (MAP) kinases was quantified as the ratio of phosphorylated protein to
the corresponding total protein detected on the same blot.
Indirect immunofluorescence microscopy
To visualize filamentous actin (F-actin), cells grown on 22 mm (no. 1)
glass coverslips were fixed with 3.7% formaldehyde in PBS for 15 minutes,
permeabilized with 0.05% Triton X-100 in PBS for 5 minutes and stained with
tetramethylrhodamine B isothiocyanate (TRITC)-phalloidin (1 µg/ml) (Sigma)
for 30 minutes. Changes in the intracellular distribution of -actinin
were assessed in formaldehyde-fixed cells using an anti-
-actinin
antibody (clone BM-75.2; Sigma) (1:200) followed by fluorescein isothiocyanate
(FITC)-conjugated goat anti-mouse antibody (Vector). Distribution of
-catenin was assessed in formaldehyde-fixed cells using an
anti-
-catenin antibody (Sigma) (1:200) followed by a
Rhodamine-Red-conjugated goat anti-rabbit antibody (Jackson ImmunoReseasrch
Laboratories, Inc). Fascin was visualized in methanol-fixed cells using
antibody 55k-2 as described previously
(Guvakova et al., 2002
). The
samples were examined under a Nikon Eclipse E600 MRC 1024 confocal
laser-scanning (Bio-Rad) microscope with a Plan Apo 60x/1.4 oil
objective lens (Nikon). Images were acquired using the Bio-Rad LaserSharp 2000
software run under the OS/2 Warp ConnectTM operating system. Images were
collected as stacks of xy optical sections (z-series) taken at different focal
planes within the sample.
Transmission electron microscopy
Cells were grown to confluence in regular culture medium on 60 mm tissue
culture dishes (Falcon). Following overnight serum starvation, cells were
either stimulated with 50 ng/ml IGF-I for 15 minutes or kept in serum-free
medium at 37°C until fixing. Cells were fixed in fresh fixative (1%
glutaraldehyde, 1% OsO4 in 0.05M phosphate buffer, pH 6.2) for 45
minutes at 4°C (Tilney and Tilney,
1994), washed three times in water at 4°C to remove phosphate,
stained with 1% uranyl acetate overnight in the dark at 4°C, dehydrated in
ethanol and 2-hydroxypropyl methacrylate and then embedded in an epon
substitute LX-112 (Ladd Research Industries Inc, VT). Sections were cut with a
diamond knife and examined with a Philips 200 electron microscope (Philips
Scientific, Mahwah, NJ). Photographs were taken of sections mounted on
uncoated grids. Images of the scanned photographs were processed as Adobe
Photoshop files for the figures presented.
Four-dimensional imaging of EGFP--actinin dynamics in live
cells
Cells grown in glass-bottomed dishes in D-MEM/F-12 containing 15 mM HEPES
buffer were placed within a chamber heated at 37°C mounted on the stage of
a Nikon Eclipse TE 300 confocal laser-scanning (Bio-Rad) microscope. Multiple
replicate fields of cells were observed over 10 minutes in serum-starved
cultures or were followed for 10 minutes in the presence of 50 ng/ml IGF-I.
For the IGF-I time course study, four-dimensional (4D) data sets (sequences of
z-series collected over time) were acquired using the acquisition function of
the LaserSharp 2000 (Bio-Rad) software. Digital confocal images were captured
in real time as a z series (z-step=0.5 µm) throughout the thickness of each
cell. The 4D series was reconstructed into a 3D projection sequence using the
LaserSharp processing function. Single sections were selected for export into
Photoshop files for the figures presented.
Time-lapse video microscopy
MCF-7, MCF-7/IGF-IR/WT or MCF-7/IGF-IR/DK cells were plated at low density
in DMEM/F-12 in Slideflasks (Nunc, Denmark) and allowed to adhere, spread over
and establish cell-cell contacts. Time-lapse video microscopy was carried out
in a 37°C environmental chamber using a Zeiss Axiovert 100 microscope
linked by a Sony SS-M37OCE CCD camera to a video recorder driven by an EOS BAC
900 animation controller. Recordings were made at a rate of 10 frames per
minute over a 60-80 minute period for each sample. Each sample contained
between 46 to 102 cells per field, and all conditions were analyzed in three
independent experiments. Alterations in cell motility and shape were measured
from traces of the video images documenting (a) paths of the cell centroid,
which were used to calculate cell velocities as movement over time; (b) the
changes in cell outline over time, from which the percentage of cells with
edge ruffles was calculated; and (c) the appearance of apical ruffles, which
appeared as dynamic phase dark ridges at the nuclear and perinuclear regions
of the apical cell membrane. In experiments involving IGF-I treatment, video
recording was started within 1 minute of adding the growth factor. A QuickTime
movie was generated from video records using the Improvision Openlab software
package.
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Results |
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To compare receptor signaling over time in transfected cells, phosphorylation of the IGF-IR and IRS-1 was examined in the wild-type and kinase-dead transfectants treated with IGF-I. Cells expressing the wild-type IGF-IR had higher tyrosine phosphorylation of both the IGF-IRß subunit and IRS-1 between 5 and 60 minutes following IGF-I treatment. By contrast, MCF-7/IGF-IR/DK cells showed a complete lack of tyrosine phosphorylation of either the IGF-IRß subunit or IRS-1 (Fig. 1b). These data establish that IGF-IR signaling is blocked in MCF-7/IGF-IR/DK cells at the first step of receptor kinase activation and IRS-1 phosphorylation.
The requirement for the IGF-IR kinase in the disorganization of the
epithelial sheet
We next compared migratory behavior of the parental MCF-7 cell line and its
derivative clones in response to IGF-I. Parental cells treated with IGF-I
became migratory and pulled apart from one another at cell-cell contacts
within colonies. Cell surfaces changed markedly, exhibiting intensive membrane
ruffling over the apex and at the cell margins. When clones were treated with
IGF-I, MCF-7/IGF-IR/WT but not MCF-7/IGF-IR/DK cells developed protrusions,
separated from one another and became motile in the direction of intense
peripheral ruffling. The MCF-7/IGF-IR/WT cells initially located at the edges
of colonies became most motile; these cells detached completely from
neighboring cells and migrated freely as single cells until they came into
contact with other cells (Movie 1; available at
jcs.biologists.org/supplemental).
To estimate the differences in cell migratory behavior in response to IGF-I, we compared ruffling activity and cell movement rates in MCF-7/IGF-IR/WT and MCF-7/IGF-IR/DK cells. Before stimulation, small zones of minor membrane ruffles at the free edges of cells were observed occasionally in both cell types, but apical membrane activity was undetectable. Stimulation with IGF-I resulted in a dramatic increase in the membrane ruffling of MCF-7/IGF-IR/WT over the period of 1 hour and that correlated temporally with cell-cell separation (Fig. 2A). 100% of IGF-I-stimulated MCF-7/IGF-IR/WT cells developed prominent apical membrane activity, and the percentage of cells with peripheral, phase dark ruffling activity doubled (Fig. 2Ba-c). The rates of cell motility were related to the initial position of cells within colonies and varied between 20.7±2.4 µm/hour (mean±s.e.m.) for cells located inside the colonies and 59.7±7.6 µm/hour (mean±s.e.m.) for cells at the periphery of colonies.
|
In sharp contrast, MCF-7/IGF-IR/DK cells showed no increase in cortical ruffling upon IGF-I stimulation, and less than 20% of MCF-7/IGF-IR/DK cells developed apical membrane protrusions (Fig. 2Bd-f). Furthermore, only 17% of the cells showed any motile activity with mean velocities 3.8±1.6 µm/hour inside the colonies and 13.9±1.9 µm/hour at the colony margins. These results established that in MCF-7 cells IGF-IR tyrosine kinase activity was essential for the formation of apical membrane projections, the increase of lateral ruffling and separation of cells followed by migration.
The IGF-IR kinase regulates concomitant reorganization of
-actinin and F-actin at cell-cell contacts
In MCF-7 cells, the activation of the IGF-IR causes reorganization of the
actin cytoskeleton (Guvakova and Surmacz,
1999). Here, we used indirect immunofluoresence to assess whether
in separating cells reorganization of F-actin in cell-cell contacts is coupled
to the distribution of
-catenin and
-actinin, two principle
actin-binding proteins that connect microfilaments to adherens junction
receptor complexes. In serum-starved MCF-7/IGF-IR/WT cells,
-catenin
and
-actinin formed continuous lines along the borders of mature
cell-cell contacts, where F-actin was also localized extensively
(Fig. 3Aa,e,i). Within 5
minutes of IGF-I stimulation, about 30-35% of cells had punctuated instead of
continuous distribution of
-catenin, which indicated the onset of the
disruption of adherens junctions. By contrast,
-actinin appeared highly
concentrated at cell margins, and the short actin-enriched projections
appeared at cell-cell contacts (Fig.
3Ab,f,j). The formation of the cortical
-actinin and
F-actin structures correlated temporally with the loosening of cell-cell
contacts and membrane ruffling documented by time-lapse video microscopy. By
15 minutes, the majority of the cells (up to 85%) had discontinuous
-catenin staining at cell-cell borders. In sharp contrast,
-actinin and F-actin continue to highly concentrate within marginal
membrane protrusions (Fig.
3Ac,g,k). At 60 minutes,
-catenin localized diffusely in
the cytoplasm and in the rare remaining cell-cell contacts, whereas
-actinin and F-actin concentrated at the edges of multiple membrane
protrusions (Fig. 3Ad,h,l).
These findings indicated that IGF-I-induced separation of MCF-7 cells was
associated with the progressive reduction of
-catenin and the increase
of
-actinin and F-actin at cell-cell contacts.
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The reduction of -catenin in cell-cell contacts can be achieved
experimentally by lowering the concentration of extracellular calcium ions.
This process is thought to be associated with unclustering and/or
internalization of Ecadherin-catenin complexes
(Kusumi et al., 1999
). When
confluent MCF-7/IGF-IR/WT cells were shifted to PBS containing 0.5 mM EDTA or
PBS free of calcium,
-catenin quickly translocated from cell-cell
contacts to the cytoplasm before dissociation of the cells. This procedure,
however, did not stimulate the assembly of cortical structures containing
-actinin and F-actin or cell motility (data not shown). We propose that
IGF-IR-mediated reorganization of the actin cytoskeleton at cell-cell contacts
is specific for the process of `active cell separation' followed by migration
as opposed to experimentally induced `passive cell dissociation' unrelated to
cell movement.
To determine the role of the IGF-IR kinase activity in active cell
separation, we analyzed the effect of IGF-I on localization of -actinin
and F-actin in MCF-7/IGF-IR/DK cells, in which the dominant-negative receptor
no longer activated IGF-IR signaling. In these cells, IGF-I treatment did not
alter localization of either
-actinin or F-actin over a 60 minute
period (Fig. 3B). The cells
continued to have apico-basal polarity and to be organized in tight patches.
Thus, in MCF-7 cells, both disorganization of cell-cell contacts and the
concomitant concentration of
-actinin and F-actin at cell peripheries
are dependent on the catalytic activity of the IGF-IR kinase.
IGF-I activates colocalization of -actinin with highly ordered
actin microspikes
To examine in detail the sites of -actinin and F-actin
colocalization before and after IGF-I stimulation, we used double
immunofluorescence staining (Fig.
4A). In serum-starved MCF-7/IGF-IR/WT cells,
-actinin
co-distributed with F-actin in a circumferential band, small cytoplasmic
aggregates and the tips of the stress fiber-like filaments
(Fig. 4Ac). Within 5 minutes of
IGF-I stimulation, F-actin appeared to be depleted from the cytoplasm and was
instead concentrated in short (
2 µm) bundles that form highly ordered
arrays of spikes at cell peripheries (Fig.
4Ad). At the same time,
-actinin showed a striking
enhancement in its colocalization with F-actin at the basal portions of actin
bundles within microspikes. Notably, there was no
-actinin in the
extremities of microspikes where F-actin persisted in the tight bundles
(Fig. 4Af). These experiments
revealed that IGF-I activates localization of
-actinin at the base of
actin microspikes organized in well ordered arrays in lateral zones of
separating cells.
|
To examine more deeply the structure of microspikes in the separating
cells, we repeated the IGF-I stimulation experiments, this time processing
cells for transmission electron microscropy (TEM). Confluent serum-starved
cells had intercellular contacts typical of polarized epithelium
(Fig. 4Ba). Within 15 minutes
of IGF-I stimulation, finger-like projections formed in the lateral zones of
the opposing cells (Fig. 4Bb).
That correlated temporally with appearance of -actinin-actin
microspikes identified by immunofluorescence. The average diameter of the
membrane-flanked individual projection was 0.24±0.03 µm
(mean±s.e.m.), the lengths of projections measured from tip to base
varied from 0.27 µm to 1.21 µm. The shorter projections were in close
contact with each other, whereas the elongated ones were separated by gaps.
Clearly, the core of each projection consisted of a strap of bundled actin
oriented perpendicularly to the cell-cell contact zones. Actin bundles
traversed projections, extended through the cell body and embedded in the
actin meshwork, which coincided well with positioning of the cortical
cytoskeleton relatively to the apical membrane and cell nucleus. The average
length of the individual actin strap was 1.94±0.15 µm
(mean±s.e.m.). That agreed well with the estimated lengths of actin
bundles visualized by immunofluorescence. Thus, the ultra-structural analysis
confirmed that lateral projections induced by IGF-I in separating cells are
actin-enriched microspikes.
In living cells -actinin microspikes appear as motile
apico-lateral structures
To study the dynamics of -actinin in living cells, we took advantage
of confocal laser-scanning microscopy and examined the spatial and temporal
effects of IGF-I on full-length
-actinin tagged with EGFP. The interior
details from multiple focal planes were recorded as they changed over time (4D
imaging). For thorough analysis of
-actinin localization, the
representative sections are shown in Fig.
5.
|
Typically, without IGF-I treatment, -actinin lined the borders of
the adjacent MCF-7/IGF-IR/WT cells. The thin band of
-actinin appeared
close to the lateral cell membranes in the most apical optical section and
through the cell thickness of 10-12 µm.
-actinin was also localized
within small aggregates scattered throughout the cytoplasm and in the fine
dotted or streak-like focal structures about 2.5 µm above the substrate
surface (Fig. 5Aa,d,g). Once
cells were stimulated with IGF-I, the aggregates of
-actinin cleared
off the cytoplasm and an increased amount of
-actinin became associated
with cell boundaries where the nascent projections were formed. Within a few
minutes, the propelling microvilli-like projections containing
-actinin
extended and retracted laterally and upward; the most elongated projections
(of 6-10 µm in length) bent across the cell apex
(Fig. 5Ab,c). The short (of 2-4
µm in length)
-actinin projections were also seen through a series
of the middle optical sections spanning 10 µm in height
(Fig. 5Ae,f). Within 5 minutes,
some cells vanished from view in the most apical sections and then from the
series of lateral sections (compare circled areas in
Fig. 5Aa with b,d and e). In
the same cells,
-actinin motile ruffles formed along the boundaries of
the basal membrane. These basal ruffles resembled growth-factor-induced
ruffles described in KB cells (Kadowaki et
al., 1986
). In parallel, the initially minute
-actinin-containing focal structures enlarged and oriented in the
direction of cell spreading (Fig.
5Ag-i, circled). By 9 minutes of IGF-I stimulation, these focal
structures appeared closer to the substratum than they were in the untreated
cells (1.5 µm compared with 2.5 µm from the substrate level). Thus, we
found that in separating living cells,
-actinin is localized in the
dynamic apico-lateral microspikes besides focal adhesions and the edges of the
basal ruffles.
In MCF-7/IGF-IR/DK cells, despite the continuous treatment with IGF-I,
-actinin remained predominantly localized to lateral membranes of cells
tightly adhered to each other. Only minor time-dependent redistribution of
-actinin occurred in the cytoplasm and at the cell borders
(Fig. 5B); these changes were
typical of all live MCF-7-derived cells regardless of IGF-I stimulation. Taken
together these observations clearly show that activation of the IGF-IR causes
a time-dependent accumulation of
-actinin-EGFP in the apico-lateral
microspikes of separating cells.
The development of -actinin mutants for molecular genetic
analysis of the requirements for
-actinin in microspikes
In the -actinin molecule, the N-terminal head contains highly
conservative actin- and phospholipid-binding sites, the central rod domain is
composed of four spectrin-like repeats implicated in dimerization of
-actinin monomers and interaction with other proteins and the
C-terminal end of the rod provides binding sites for ions of calcium as well
as proteins (Hemmings et al.,
1992
; Matsudaira,
1994
; McGregor et al.,
1994
; Fukami et al.,
1996
). The characterization of
-actinin mutants has been
particularly difficult in non-muscle cells owing to the rapid lethality of
cells expressing the mutant proteins (Scultheiss et al., 1992;
Hijikata et al., 1997
). To
define the contribution of individual domains to the function of
-actinin in microspikes, we have constructed new pEGFP N1-vector-based
plasmids encoding the mutant
-actinins as C-terminal chimeras with EGFP
(Fig. 6A), and that allowed us
to monitor fusion proteins soon after transfection. 230 amino-acid residues in
the N-terminus were deleted to yield a mutant
-actinin lacking the
entire actin-binding region (
N). To inactivate
-actinin in the
C-terminus (
C), the last 168 amino acids were removed (described in the
Materials and Methods).
|
MCF-7/IGF-IR/WT cells were transfected with plasmid DNA encoding either
control EGFP or EGFP chimeras with a full-length wild-type (WT), mutant
N and
C
-actinin. Cell extracts were resolved
electrophoretically, and proteins transferred on the membrane were probed
sequentially with antibody to GFP and
-actinin. Western immunoblotting
has confirmed protein expression and the expected molecular weights of
exogenous EGFP (27 kDa), WT (127 kDa),
N (104 kDa) and
C (109
kDa) EGFP-
-actinins (Fig.
6B).
Both head and tail domains of a-actinin are required for the
development of highly ordered microspikes
To visualize the dynamics of -actinin-EGFP fusion proteins specific
to the regions of cell-cell contacts, we used confocal laser-scanning
microscopy and collected stacks of images from the middle optical sections
crossing nuclei. In serum-starved cells with mature cell-cell contacts, only
the wild-type and
C
-actinins incorporated into pre-existing
cell-cell junctions. Unlike the wild-type and
C
-actinin,
N
-actinin did not associate with the circumferential actin ring
and was instead localized diffusely throughout the cell body
(Fig. 6Ca-c).
In WT -actinin transfectants, IGF-I triggered the expected
reorganization of cell-cell contacts and the assembly of
-actinin-actin
microspikes in the lateral zones (Fig.
6Cd).
N
-actinin did not colocalize with actin
microspikes and remained diffused throughout the cell body
(Fig. 6Ce). The number of
C
-actinin-transfected cells with
-actinin-actin spikes
was half that of wild-type transfectants. Moreover, the spatial relationship
between
C
-actinin and F-actin was disturbed, as microspikes did
not organize into well ordered arrays (Fig.
6Cf). In two independent experiments, the appearance of
microspikes was assessed quantitatively
(Table 1). Collectively, these
results demonstrate that in the
-actinin molecule both the head and
tail regions are required for the function of microspikes. The N-terminal part
of
-actinin is necessary for association of
-actinin with actin
bundles, whereas the C-terminal tail of the rod is needed for correct assembly
and/or positioning of microspikes at cell peripheries.
|
The mechanism of active cell separation and microspike assembly
requires activities of PI 3-kinase-generated phospholipids
To address the role of IGF-IR kinase signaling in regulating microspike
assembly in relation to cell separation, we used four specific pharmacological
inhibitors of PI 3-kinase and MAP kinase signaling pathways
(Fig. 7). They are LY 294002, a
specific inhibitor of the catalytic subunit of the PI 3-kinase; a D3-modified
phosphatidylinositol (PI) ether lipid analog (also called Akt inhibitor); PD
98059, a synthetic inhibitor of the MAP kinase-activating enzyme, MAPK/ERK
kinase (MEK1); and UO 126, an inhibitor of MEK1 and MEK2. In MCF-7/IGF-IR/WT
cells treated with LY 294002, the normal induction of phosphorylation of the
PKB/Akt by putative PDK2 on serine 473 was downregulated by 90% and 84% and by
3-phosphoinositide-dependent kinase 1 (PDK1) on threonine 308 by 28% and 20%,
and at 5 and 15 minutes, respectively. The 3D-modified PI-analog also
downregulated signaling associated with the activities of PI
3-kinase-generated phospholipids, although its effect on phosphorylation of
PKB/Akt was much less. The relative phosphorylation of PKB/Akt on serine 437
was reduced by 8% and 19%, and on threonine 308 by 9% and 11% at 5 and 15
minutes of IGF-I stimulation (average of five experiments)
(Fig. 7a). PD 98059 reduced the
relative phosphorylation of ERK1/2 MAP kinases on threonine 202 and tyrosine
204 by 73% and 67% at 5 and 15 minutes of IGF-I treatment (average of four
experiments). UO 126 was a much more potent inhibitor of ERK1/2 MAPK kinases;
it blocked completely IGF-I-stimulated and basal phosphorylation of these
kinases (Fig. 7b and data not
shown).
|
Pretreatment of MCF-7/IGF-IR/WT cells with 10 µM LY294002 and 40 µM
PI analog before the addition of 50 ng/ml IGF-I blocked the formation of
cortical microspikes and cell separation in response to IGF-I [compare
Fig. 8A,b with c,d also with
Guvakova et al. (Guvakova et al.,
2002)]. LY 294002- and PI-analog-pretreated cells remained
organized in tight colonies during at least 60 and 30 minutes of the IGF-I
treatment, respectively. By contrast, there was no apparent inhibitory effect
of 50 µM PD 98059 on cell separation; cells, exposed to this drug or not,
appeared spindle-shaped, separated from each other and migrated within 60
minutes of IGF-I stimulation. The formation of well ordered
-actinin-actin microspikes was slightly disturbed by PD 98059 (compare
Fig. 8A,b with e). The more
potent inhibitor of MEK1 and MEK2, UO 126, did not prevent IGF-stimulated
concentration of
-actinin and actin in the peripheral microspikes
(Fig. 8Af); however, it
inhibited cell migration radically within first 60 minutes of IGF-I
stimulation (data not shown).
|
MEK1/2 signaling downstream of the IGF-IR kinase is essential for
migration of separated cells
The above findings prompted our suggestion that MEK1/2 signaling is
unnecessary for initial steps of cell-cell separation but may be essential for
movement of the cells separated in response to PI 3-kinase signaling. To test
this idea, we compared the effect of the PI analog and UO 126 on
IGF-stimulated reorganization of cell-cell contacts, this time monitoring
distribution of -catenin, a molecular marker of adherens junctions, and
fascin, a marker of the peripheral actin microspikes. In contrast to
-actinin, fascin localizes along the length of actin bundles induced by
IGF-I and that allows visualizing microspikes without co-staining for F-actin
(Guvakova et al., 2002
).
Pretreatment of cells with the PI analog blocked IGF-I-stimulated
redistribution of
-catenin and thereby dissolution of adherens
junctions. This inhibitor also blocked the formation of microspikes containing
fascin (Fig. 8Ba,c; inset in c,
fascin microspikes induced by IGF-I). In sharp contrast, total inhibition of
MEK1/2 activity by UO 126 did not prevent a loss of
-catenin from
adherens junctions and did not block the formation of fascin microspikes at
cell peripheries (Fig. 8Bb,d).
Strikingly, inhibition of MEK1/2 by UO 126 had a marked effect on stress
fibers. Normally, activation of the IGF-IR in MCF-7 cells results in a rapid
disassembly of stress fibers followed by re-assembly of microfilaments after
15 minutes of IGF-I stimulation. The latter coincides with advancing long
protrusions and moving the cell body [seen in
Fig. 3Ai-l and described in
detail in (Guvakova and Surmacz,
1999
)]. Pretreatment with UO 126 had no effect on disassembly of
stress fibers; however, it prevented their re-assembly as well as the
development of the long membrane protrusions after 15 minutes of IGF-I
stimulation (compare Fig. 8Ca with
b).
We conclude that separation of MCF-7 breast cancer cells in response to IGF-I is dependent on signaling from the activated IGF-IR through the PI 3-kinase and its phospholipid products, and that signaling through MEK1/2 may be necessary for subsequent cell locomotion.
![]() |
Discussion |
---|
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---|
Cell motile responses mediated by the IGF-IR
It has previously been established that polypeptide growth factors acting
through receptor tyrosine kinases can induce transition of cells from an
epithelial to a motile fibroblastic phenotype, a phenomenon that resembles
epithelial-mesenchymal transition, which occurs during development
(Boyer and Thiery, 1993). IGF-I
is mainly known as a mitogenic growth factor, and signaling through the IGF-IR
in mammary epithelial cells has been chiefly studied in respect to effects on
cell proliferation and apoptosis (reviewed by
Lee et al., 1998
;
Chernicky et al., 2000
).
Nevertheless, a role for the IGF-IR in motility of breast carcinoma cells had
been proposed more than a decade ago on the basis of the results of the Boyden
chamber assay (Kohn et al.,
1990
). It should be noted, however, that in this assay cells are
artificially separated from each other before examination, and it is therefore
not possible to analyze mechanisms of epithelial sheet disintegration.
In this study we examined in detail the effects of IGF-I on motile behavior
of cells within an epithelial sheet. We have now demonstrated that, like other
growth factors with scattering function, IGF-I induced cell motile responses
in a biphasic manner, first causing separation and subsequently causing
migration of the cells. The phase of active separation lasted only for 15-30
minutes and was unusually short compared with the 5-16 hours reported for
HGF/SF, FGF-1 and EGF (Stoker and
Perryman, 1985; Savagner et
al., 1997
; Müller et al.,
1999
). During the separation phase, IGF-I stimulated highly
dynamic activity of the entire cellular membrane: formation of apical ruffles
and motile apico-lateral microspikes, dissolution of adherence junctions and
an increase in basal membrane ruffling. Although the precise mechanism of such
rapid morphological alterations is unknown, the appearance of dynamic
microspikes at the cell-cell contacts and temporal correlation of this event
with disintegration the adherens junction complex led us to suggest that
microspikes facilitate cell-cell detachment. In addition to acute cell
separation, video tracking has revealed uncommonly fast rates of migration of
single MCF-7/IGF-IR/WT cells. Collectively these data establish for the first
time that activation of the IGF-IR can in the short-term cause destabilization
of cell-cell interactions, leading to quick separation and migration of breast
cancer epithelial cells.
IGF-IR-mediated active cell separation: structural
reorganizations
EGF modulates the interaction between the adherens junction receptor
E-cadherin and the actin cytoskeleton, although associated cytoskeletal
reorganizations have not been characterized
(Hazan and Norton, 1998). Our
previous work has shown that in MCF-7 cells, IGF-I induces rapid disassembly
of stress fibers and extensive reorganization of the cortical actin
(Guvakova and Surmacz, 1999
).
We have now demonstrated that disassembly of the cortical actin belt coincides
with mobilization of
-actinin and actin into apicolateral microspikes
and displacement of
-catenin from the adherens junctions. This agreed
with the recent report that activation of the IGF-IR induces redistribution of
E-cadherin and ß-catenin from the adherens junctions into the cytoplasm
(Morali et al., 2001
).
What drives -actinin into cell-cell contacts? As an actinbinding
protein, intact
-actinin shows a prominent colocalization with
filamentous actin. Yet
-actinin can bind to a number of molecules, in
addition to actin (Crawford et al.,
1992
; Otey et al.,
1993
; Kroemker et al.,
1994
; McGregor et al.,
1994
; Christerson et al.,
1999
) that perhaps determine the localization of
-actinin.
For example, in Cos and PtK2 cells, mutated
-actinins with a deleted
actin-binding site were not incorporated into stress fibers, although they
localized to ruffled membranes and adherens junctions
(Hemmings et al., 1992
;
Hijikata et al., 1997
). The
prominent localization of
-actinin to adherens junction has been
related to the direct association between the central rod domain of
-actinin and the C-terminus of
-catenin
(Knudsen et al., 1995
;
Nieset et al., 1997
). In
epithelial culture,
-actinin colocalizes with vinculin and the
cadherin-catenin complex. In this study, we used GFP-labeled
-actinin
to follow distribution of
-actinin in living cells before and after
IGF-I stimulation. Our results with a GFP-labeled mutant of
-actinin,
in which the
-catenin-binding site was preserved while the
actin-binding domain was removed, support the idea that both
-catenin
and actin-binding sites of
-actinin are essential for localization of
this protein into the mature cell-cell junctions. The interaction of the
C-terminal end of
-actinin with vinculin does not seem to be required
because a GFP-tagged
C
-actinin mutant in which the
vinculin-binding site was disrupted did not interfere with the ability of
-actinin to localize to intercellular junctions. The development of
microspikes in response to IGF-I required at least two fully functional
regions of the
-actinin molecule. As expected, the head of
-actinin was obligatory for binding to actin spikes. The C-terminal
tail of
-actinin was required for correct assembly and/or stabilization
of microspikes because the truncation of this region reduced the number of
-actinin/actin microspikes and caused their misalignment at the cell
periphery. According to the previous suggestion, it is possible that the
actin-binding activity of
-actinin requires a ternary interaction with
the C-tail as modifications in the EF-hand domain of
-actinin have
deleterious effects on the activity of its actin-binding domain
(Witke et al., 1993
;
Dubreuil and Wang, 2000
).
Additionally, direct interaction of
-actinin with vinculin, which is
probably disturbed in the
C
-actinin mutant, might be required
for correct organization of microspikes. Thus our findings establish that
functional
-actinin is necessary for the development of microspikes and
is not passively reorganized following actin.
Our results, while supporting previous findings on IGF-I-stimulated actin
polymerization at the cell periphery
(Kadowaki et al., 1986;
Isumi et al., 1988
), further
demonstrate that activation of the IGF-IR coordinates reorganization of actin
into peripheral microspikes. Localization of
-actinin at the base
rather than at the tips of microspikes implies that other actin-crosslinking
protein(s) compose the core of a spike. A good candidate for this role is the
55 kDa actin-bundling protein fascin, which acts as a crosslinker, causing
aggregation of actin into tight bundles
(Edwards and Bryan, 1995
;
Kureishy et al., 2002
). In
vitro, fascin and
-actinin synergistically affect mechanical properties
of actin filaments (Tseng et al.,
2001
). In vivo, fascin plays a role in extending the membrane
during cell spreading and migration, and overexpression of fascin in pig
epithelial cells caused the disorganization of cell-cell contacts
(Yamashiro et al., 1998
).
Fascin also acts as a negative regulator of cell-cell interactions in rat
mammary epithelial cells (Wong et al.,
1999
). Activation of the IGF-IR in MCF-7 cells induces rapid
relocalization of fascin from the cytoplasm to the peripheral actin
microspikes (Guvakova et al.,
2002
). We reason now that the activated IGF-IR induces cortical
actin dynamics beneath the plasma membrane by increasing actin polymerization
and promoting crosslinking of actin by fascin to stabilize the cores of
microspikes and by
-actinin to properly position these nascent
projections at the cell periphery. Although the significance of microspikes in
cell-cell separation remains hypothetical, one possibility is that microspikes
play an active cytomechanical role in the destabilization of interactions
between opposing cells. Alternatively, microspikes may be the form of
cell-cell contacts that are transient and more flexible than adherens
junctions and therefore provide the intermediate strength of adhesion between
separating cells.
IGF-IR-regulated cell motility: signaling pathways
Cell-cell detachment and microspike formation was completely blocked in
MCF-7/IGF-IR/DK cells, clearly indicating the direct causative relationship
between IGF-IR activation and -actinin redistribution. Although it is
conceivable that IGF-IR signaling affects the phosphorylation status of
-actinin, at least two lines of evidence imply that tyrosine
phosphorylation of
-actinin is not relevant to the development of
microspikes in MCF-7 cells. First, there was no detectable difference in the
tyrosine phosphorylation of
-actinin immunoprecipitated from cells
stimulated with IGF-I (see Materials and Methods). Second, the WT
-actinin-EGFP chimera, which lacked the only identified phopshotyrosine
residue 12 within the amino-acid motif QTNDY
(Izaguirre et al., 2001
), was
nevertheless functionally active and localized to microspikes.
As relocalization of -actinin into cortical structures was detected
after 1-2 minutes of IGF-I stimulation, we predicted that the early signaling
triggered by the ligand-receptor interaction promotes the development of
microspikes. In MCF-7 cells, activation of the IGF-IR kinase quickly drives
the formation of a complex between the receptor and substrates IRS and Shc
that brings about activation of the downstream cascades of serine/threonine
kinases and the PI 3-kinase. Interestingly, in different epithelial cells,
different kinases play roles in cell motility. In MDCK canine epithelial
cells, activation of both the PI 3-kinase and MAP kinase was found to be
essential for the motile response to HGF/SF
(Royal and Park, 1995
;
Potempa and Ridley, 1998
). The
function of MAP kinases was delineated in scattering triggered by HGF/SF in
HT29 colon carcinoma cells and haptotaxis in FG carcinoma cells and T47D
breast carcinoma cells (Klemke et al.,
1997
; Herrera,
1998
; Spencer et al.,
2000
), whereas pretreatment with the MEK1 inhibitor, PD 98059, was
dispensable for the chemotactic response of MCF-7 cells to IGF-I
(Manes et al., 1999
). Others
have reported that IGF-I stimulates colonic epithelial cell migration in a
wound healing assay through multiple signaling pathways including PI 3-kinase,
MAP kinases and PKC-
and -
(Andre et al., 1999
). In MCF-7
cells expressing the dominant-negative receptors, activity of the PI-3 and the
ERK1/2 MAP kinases is downregulated via markedly reduced signaling through
IRS-1 and Shc pathways (in this paper and M.A.G., unpublished). To dissect
signaling pathways essential for cell motility, we applied selective
pharmacological inhibitors of PI 3-kinase and MEK. The MEK inhibitor also
inhibits ERK1/2 MAP kinases (summarized in
Fig. 9a). Using
immunofluorescence staining coupled with biochemical analysis, we found that
the activity of PI 3-kinase rather than that of the MAP kinases was required
for disassembly of adherens junctions and redistribution of actin,
-actinin and fascin into microspikes during cell separation.
Subsequently, the activity of MEK1/2 appeared to be necessary for re-assembly
of stress fibers, which may be involved in the extension of cell protrusions
and the translocation of the cell body (model in
Fig. 9b). As activities of the
ERK1/2 MAP kinases may enhance myosin light chain kinase (MLCK) activity,
leading to the phosphorylation of myosin light chains (MLC)
(Klemke et al., 1997
), it
seems possible that IGF-IR signaling sequentially coordinates cell-cell
separation and phospho-MLC-dependent cytoskeletal contraction culminating in
cell movement.
|
The function of the PI 3-kinase targets implicated in actin reorganization
is dependent on PI 3-kinase-generated phospholipids
(Nobes et al., 1995).
Moreover,
-actinin may bind to the PI kinase and its product,
phosphatidylinositol (3,4,5)-trisphosphate
[PtdIns(3,4,5)P3]
(Shibasaki et al., 1994
;
Greenwood et al., 2000
). To
investigate the role of PI-3-kinase-generated phosphatidylinositides in cell
separation, we used a D3-modified PI analog, a competitive inhibitor of the PI
3-kinase with respect to PI, synthesized to selectively block the effect of
myo-inositol-derived second messengers of the PI 3-kinase
(Hu et al., 2000
). This
compound prevented a loss of
-catenin from adherens junctions as
prevented redistribution of
-actinin and fascin into actin microspikes,
suggesting a role for D3 phospholipids in IGF-I-stimulated cell separation.
Although PKB/Akt possesses a pleckstrin-homology (PH) domain and may bind to
PI-3-kinase-generated phospholipids, its phosphorylation in MCF-7 cells was
only slightly reduced by the PI analog. Thus, the precise role of this kinase
as well as other proteins bearing PH domains, including several adaptor
proteins and GTP/GDP exchange and GTP-activating proteins for the ARF/Rho/Rac
family of GTP-binding proteins, has yet to be investigated.
In summary, we have identified the first biochemically defined pathway in
which the IGF-IR tyrosine kinase, through activities of the PI 3-kinase and
-actinin, promotes active separation in human breast cancer cells. We
have also established the requirement for MEK1/2 activity in IGF-IR-mediated
movement of these cells. Further development of
-actinin mutants and
the measurements of the direct association between
-actinin and other
molecules in living cells should make it possible to understand the dynamic
regulation of microspikes and their role in cell separation. Further studies
are needed to reveal the precise mechanisms by which MAP kinases control
motility in the separated breast cancer cells.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
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