* Departments of Medicine and Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI 53706; and Department of Experimental Pathology, Lund University, 22185 Lund, Sweden
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Abstract |
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1A integrin subunits with point mutations
of the cytoplasmic domain were expressed in fibroblasts derived from
1-null stem cells.
1A in which
one or both of the tyrosines of the two NPXY motifs
(Y783, Y795) were changed to phenylalanines formed
active
5
1 and
6
1 integrins that mediated cell adhesion and supported assembly of fibronectin. Mutation
of the proline in either motif (P781, P793) to an alanine
or of a threonine in the inter-motif sequence (T788) to
a proline resulted in poorly expressed, inactive
1A.
Y783,795F cells developed numerous fine focal contacts and exhibited motility on a surface. When compared with cells expressing wild-type
1A or
1A with
the D759A activating mutation of a conserved membrane-proximal aspartate, Y783,795F cells had impaired ability to transverse filters in chemotaxis assays.
Analysis of cells expressing
1A with single Tyr to Phe
substitutions indicated that both Y783 and Y795 are
important for directed migration. Actin-containing microfilaments of Y783,795F cells were shorter and more
peripheral than microfilaments of cells expressing wild-type
1A. These results indicate that change of the
phenol side chains in the NPXY motifs to phenyl
groups (which cannot be phosphorylated) has major effects on the organization of focal contacts and cytoskeleton and on directed cell motility.
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Introduction |
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INTEGRINS are transmembrane heterodimers that
mediate organization of focal contacts, actin-containing cytoskeleton, and extracellular matrix (1, 11, 24,
35, 56, 70). Intracellular signals cause changes in integrin
extracellular domains and thereby regulate affinity for extracellular ligands (22, 30, 33, 35, 38, 59). Regulation of the
interaction of integrins with extracellular ligands allows
control of cell adhesion and migration. Adhesion and migration, in turn, are important determinants of cell proliferation and differentiation (21, 24, 27, 34, 35, 39). Integrins
are involved in a number of pathological processes (1, 30,
35, 56, 64). Therefore, a detailed understanding is needed
of the complex "inside-out" and "outside-in" signal transduction pathways underlying and provoked by the association of integrins with extracellular ligands and intracellular
effector molecules and how these pathways control cellular behavior.
Three regions of the A splice form of 1 subunit (
1A)
cytoplasmic domains have been implicated in the function
of
1A integrins (see Fig. 1). The Asp of the membrane-proximal sequence KLLXXXXD likely forms a salt bridge
with a conserved Arg of the
subunit, thus stabilizing a
default inactive conformation (32). The other regions are
comprised of two NPXY motifs (54). These motifs are
found in the cytoplasmic domains of
1A and
1D,
2,
3,
5,
6, and
7 (see Fig. 1) (25, 49). In all except
5, the
motifs are separated by an intervening sequence of eight
residues. Substitution of a Pro for a Ser in the intervening
sequence of
3 is associated with Glanzmann thrombasthenia, a bleeding disorder caused by abnormal function of
platelet
IIb
3 (16, 17). Mutagenesis of the Asn or Pro
residues of the NPXY motifs results in
subunits that do
not localize to focal contacts (19, 54). Such changes likely alter the tight
-turn predicted for such sequences (3, 28). When the cDNA for
1A was first sequenced, the tyrosine
residues of the NPXY motifs were identified as potential
sites of phosphorylation (62). Subsequent studies demonstrated tyrosine phosphorylation of
1 in transformed cells
and of
3 in activated and aggregated platelets and in
v
3-expressing K562 cells (8, 9, 31, 40, 63). Tyrosine
phosphorylated
1 of src-transformed cells, detected by
anti-phosphopeptide antibodies, localizes to podosomes rather than focal contacts (37). Transfected
1As with one
or both Tyr mutated to Glu, Ala, or Ser localize less well
than wild-type
1A to focal contacts (19, 54). Transfected
lA with conservative mutations of Tyr to Phe, which cannot be phosphorylated, localizes normally to focal contacts
(19, 29, 48, 54, 65).
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We have studied the effects of the Tyr to Phe substitutions in the NPXY motifs on cell behavior. Mutated 1A
was expressed in mouse GD25 cells, which lack
1 integrin
heterodimers because of targeted knockout of the
1 gene
(23, 67). The Tyr to Phe mutations resulted in
1As that
were active in GD25 cells as assessed by antibody binding
and fibronectin matrix assembly assays. However, the Tyr
to Phe mutations were associated with loss of ability of
cells to undergo directed migration.
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Materials and Methods |
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Mutation of 1A
Mutant 1A constructs were generated from pBS
1A encoding full-length mouse
1A integrin subunit (67). The
1A was cloned into
pGEM7Zf by the unique XbaI and Acc65I restriction sites. Mutants were
generated by oligonucleotide-primed DNA synthesis using the U.S.E. mutagenesis kit (Pharmacia Biotech Sevrage, Uppsala, Sweden). Regions
spanning the Pm1I and Acc65I sites were analyzed by DNA sequence
analysis. Mutagenized PmlI-Acc65I fragments were isolated and ligated
into PmlI-Acc65I-digested pBS
1A to generate cDNAs encoding full-length
1A polypeptides containing the individual point mutations in the
cytoplasmic tail. The plasmid was linearized with XbaI and transfected
into
1-deficient GD25 cells by electroporation. After 72 h, selection with
5 µg/ml puromycin was started. Surviving clones were isolated and expanded. Clones stably expressing
1A were identified for each mutation.
Clones were monitored for expression of
1A by flow cytometry. If necessary, the population of higher expressing cells was selected by fluorescence detected cell sorting.
Flow Cytometry
Cells were harvested and suspended in PBS containing 3% (wt/vol) BSA.
Approximately 1.0 × 106 cells were incubated with primary antibody, and
then treated with FITC-conjugated secondary antibody at 4°C. We confirmed by titration that the dilutions of antibodies used were saturating and gave maximal specific signals. Cells (8,000 per sample) were analyzed
in a FACScan® flow cytometer (Becton and Dickinson Co., Mountain
View, CA). Monoclonal antibodies 9EG7 to mouse 1 (4, 42), MFR5 to
mouse
5, H9.2B8 to mouse
v, and 2C9.G2 to mouse
3 were all from
PharMingen (San Diego, CA). Antibody MB1.2 to mouse
1 and GoH3
to
6 were provided by Dr. B. Chan (University of Western Ontario, Ontario, Canada) and Dr. A. Sonnenberg (The Netherlands Cancer Institute, Amsterdam, Netherlands), respectively. Possible upregulation of the
9EG7 epitope by Mn2+ or recombinant adhesive modules III7-10 of fibronectin (41) was assessed by flow cytometry (4).
Metabolic Labeling and Immunoprecipitation
The cells were given methionine-free DMEM, then L-[35S]methionine
(100 µCi/ml medium) (Dupont NEN, Boston, MA) and incubated for various time periods. After incubation, conditioned media were collected,
and cells were solubilized with nonionic detergent buffer (1% [vol/vol]
Triton X-100, 0.05% [vol/vol] Tween 20, 150 mM NaCl, 2 mM PMSF, 5 µg/ml leupeptin, 0.1 µg/ml pepstatin A, 0.4 mM pefabloc SC, and 20 mM
Tris-HCl, pH 7.4). Extracellular matrix proteins were immunoprecipitated
with rabbit antibodies from metabolically labeled, conditioned media as
previously described (58). 1A integrins were immunoprecipitated from
cellular lysates with MB1.2 or 9EG7 by an established protocol (60). The
amounts of antibodies used were sufficient to precipitate all of the target antigen.
Cell Adhesion Assay
Cell adhesion assays were performed as previously described (15). Vitronectin (7), fibronectin (43), or laminin-1 (Sigma Chemical Co., St. Louis, MO) were coated at 2-10 µg/ml onto wells of a 96-well plate, and the wells were blocked with 2% BSA in PBS containing Ca2+ and Mg2+. The cells were then allowed to attach to wells for 60 min. After adhesion, nonadhered cells were removed by washing, and adhered cells were quantified by colorimetric detection at 595 nm after staining with 1% bromphenol blue.
Binding Assays
Binding of iodinated or FITC-labeled human plasma fibronectin or the 70-kD NH2-terminal fragment were assayed as described (43, 72).
Immunofluorescence
Immunofluorescence studies were performed as described previously
(57). Cells were seeded in serum-containing medium and grown on glass
coverslips for 3 d. Alternatively, cells were seeded onto glass coverslips
coated with vitronectin, fibronectin, or laminin-1 and incubated in DME
containing 0.2% BSA for 4 h at 37°C. For double staining of fibronectin
and 1 integrin, cells were incubated with FITC-labeled fibronectin in the
presence of 1-oleoyl-lysophosphatidic acid (LPA)1 (Avanti Polar Lipids,
Alabaster, AL) for an additional 1 h at 37°C. Cells were then fixed with
3.5% (wt/vol) paraformaldehyde in phosphate buffer, pH 7.4. Before
staining of intracellular antigens, the cells were permeabilized with 0.2%
Triton X-100 in TBS. After incubation with 3% BSA in TBS to block nonspecific protein binding, the fixed cells were incubated with rabbit antisera to FITC or fibronectin, and/or monoclonal antibody to paxillin, vinculin,
or
1, and then treated with FITC anti-mouse IgG, FITC anti-rabbit IgG,
lissamine rhodamine B sulfonyl chloride (LRSC)-labeled anti-rat IgG, or
rhodamine-conjugated phalloidin (Molecular Probes, Inc., Eugene, OR).
Monoclonal antibodies against human vinculin (clone hVIN-1) and chick
paxillin were from Sigma Chemical Co. and Transduction Laboratories
(Lexington, KY), respectively.
1 integrin was identified with antibody
MB1.2. FITC- or LRSC-conjugated, affinity-purified secondary antibodies were from Jackson ImmunoResearch Labs. Inc. (West Grove, PA).
Controls were done to insure the species specificity of the secondary antibodies. Photographs were taken with an Olympus BX-60 epifluorescence
microscope using additional emission filters that allowed the FITC and
LRSC or rhodamine fluorochromes to be visualized individually or simultaneously (Chroma Technology, Brattleboro, VT).
Cell Migration
Cell migration assays were performed in modified Boyden chambers containing Nucleopore polycarbonate membranes (5-µm pore size; Costar Corp., Cambridge, MA). The filters were soaked overnight in a 10 µg/ml solution of vitronectin, fibronectin, or laminin-1 or 100 µg/ml gelatin, briefly rinsed with PBS containing 0.2% BSA, air dried, and then placed in the chamber. EGF or PDGF at concentrations of 3-100 ng/ml in DME containing 0.2% BSA was added to the lower compartment of the chambers. Cells suspended in DME containing 0.2% BSA were introduced into the upper compartment of the chamber. The chambers were then incubated for 6 h at 37°C. The filters were fixed and stained, and the cells that had migrated to the lower surface were counted at 400× magnification. Two areas from each of two cells were counted. Values are the mean ± SD of cells per 0.16-mm2 field. EGF and PDGF were from Upstate Biotechnology (Lake Placid, NY) and R&D Systems, Inc. (Minneapolis, MN), respectively.
Cell Motility
The cell motility assay was adapted from an established method (50). Vitronectin, fibronectin or laminin-1, 10 µg/ml, was coated onto wells of a 96-well plate, and the wells were blocked with 2% BSA in PBS containing Ca2+ and Mg2+. Yellow polystyrene monodispersed particles (1.0-µm-diam polybeads; Polysciences, Inc., Warrington, PA) were suspended with DME, sonicated three times, and then added to wells. The plates were centrifuged at 1,400 rpm for 25 min and incubated overnight at 37°C. Cells (500 cells/well) suspended in DME containing 0.2% BSA in the absence or presence of motility factors, PDGF or EGF, 3-100 ng/ml, were introduced into wells and incubated for 16 h at 37°C. After incubation, the contents of the wells were fixed with 10% (vol/vol) glutaraldehyde, wells were washed, and clearing of beads by motile cells was assessed by phase microscopy.
Presentation of Results
Experiments were done on at least three separate occasions over an 8-mo
period on clones of cells selected to give maximal expression in the case of
the P781A, T788P, P793A, or P781,793A mutants or expression similar to
GD25-1A cells in the case of the D759A, Y783F, Y795F, or Y783,795F
mutants. The cell migration assays and microscopic analysis of microfilament bundles and focal contacts were repeated on independently derived
clones of D759A and Y783,795F cells. The experiments and photomicrographs shown were chosen as representative of differences constantly observed among cells expressing the different
1As.
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Results |
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We characterized the effects of Tyr to Phe mutations in
the NPXY motifs of 1A in mouse GD25 cells, which lack
expression of
1 integrin heterodimers because of targeted knockout of the
1 gene (23, 67). We also created
point mutations that would be expected, on the basis of
previous studies described in the Introduction, to enhance
or interfere with integrin activity (Fig. 1).
Analysis of Integrins and Cell Attachment
Expression of cell surface 1A and associated
5 and
6
subunits was analyzed by flow cytometry (Fig. 2). Transfection of
1A mutants resulted in appearance of cell surface
5 and upregulation of cell surface
6, as previously
reported for GD25-
1A cells expressing wild-type
1A
(23, 67). Binding of monoclonal antibody 9EG7, which
recognizes an extracellular epitope of
1 that can be upregulated by Mn2+ or ligand, was compared with the binding of a second anti-
1 monoclonal antibody, MB1.2, as an
index for conformationally active integrins. For GD25-
1A cells and cells expressing D759A, Y783F, Y795F, or
double Y783,795F mutant
1A, fluorescence resulting from
binding of 9EG7 was 80-100% of that due to MB1.2 binding. Attempts to upregulate the 9EG7 epitope further with
Mn2+ or recombinant adhesive modules III-7-10 of fibronectin failed (not shown). For each of the four mutants, cell
surface
1A was expressed at a level comparable to that of
GD25-
1A cells (67) in at least 6 of the ~50 clones tested.
Metabolic labeling and immunoprecipitation analysis confirmed that the production level of the D759A or Y783,795F
mutant
1A was not different from production of wild-type
1A in the GD25-
1A cells (not shown).
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Binding of 9EG7 to cells expressing P781A, T788P,
P793A, or double P781,793A mutant 1As, in contrast,
was <50% of MB1.2 binding (not shown). In addition, we
were unable to find cells expressing P781A, T788P, P793A
or double P781,793A mutant
1As that bound MB1.2 at
>30% of the expression level on GD25-
1A cells despite testing ~50 (P781A and P793A) or ~100 (T788P and
P781,793A) clones. The flow cytometry results, therefore,
indicate that wild-type
1A and the D759A, Y783F, Y795F,
and double Y783,795F mutant
1As are all expressed in
an active state by GD25 cells. In contrast, the P781A,
T788P, P793A, and double P781,793A mutant
1As are expressed less well and in a less active state.
Transfection and selection of D759A or double Y783,795F
mutant 1A resulted in variable downregulation of cell
surface
v and
3 when compared with the parent GD25
cells (not shown). Similar results were obtained previously
using surface iodination of GD25 and GD25
1A cells (67).
Cell adhesion studies were carried out to characterize
further the activity of the mutant integrins. Cells transfected with any of the set of 1As that resulted in higher
ratios of 9EG7/MB1.2 binding adhered vigorously to laminin-1, whereas untransfected GD25 cells or GD25 cells
transfected with the P781A, T788P, P793A, or P781,793A
mutant attached and spread poorly on cell culture plastic
coated with laminin-1 (Fig. 3). Adherence to laminin-1 was blocked by antibody GoH3 to
6
1 (not shown).
GD25 cells or each of the transfectants attached and
spread on vitronectin or fibronectin (Fig. 3), consistent
with previous studies showing that adhesion of GD25 cells
to fibronectin can be mediated by either the
v
3 vitronectin receptor or the
5
1 fibronectin receptor (23,
67). These results indicate that substitution of Ala for Pro
in the NPXY motifs or the thrombasthenia-like Thr-to-Pro substitution in the sequence between the two motifs results in poorly expressed integrins with little or no
6
1-mediated cell adhesive activity for laminin-1. Integrins
formed by
1A carrying the D759A mutations or substitution of Phe for Tyr in the NPXY motifs, in contrast, were
at least as active as those formed by wild-type
1A.
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Fibronectin Matrix Assembly by Cells Expressing
Mutant 1A
Fibronectin assembly is enhanced by experimental manipulations that activate 1 or
3 integrins, including activating antibodies, truncation of the
subunit cytoplasmic domain, and mutation of the membrane-proximal Asp to
disrupt the interaction between
and
cytoplasmic domains (32, 69). To learn whether expression of
1As with
Tyr to Phe mutations in the NPXY motifs also results in
hyperactive integrins, we studied assembly of fibronectin
by monolayers of transfected cells. After 3 d in 10% FBS,
fibronectin was deposited in a fibrillar pattern around
most cells expressing wild-type
1A or D759A or double
Y783,795F mutant
1A (Fig. 4). Fibronectin deposition
was more patchy in cultures of GD25 cells in that foci of
cells had copious matrix whereas nearby cells had none
(Fig. 4). No differences were found between GD25 cells and GD25 cells expressing wild-type
1A or among cells
expressing wild-type or mutant
1As in the secretion of fibronectin, or of laminin-1 or tenascin-C, when media of
cells incubated with [35S]methionine were analyzed by immunoprecipitation (not shown).
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We also analyzed fibronectin matrix deposition in short-term experiments. Fig. 5 shows fluorescence due to deposition over a 1-h period of FITC-labeled plasma fibronectin
added to cells in plates coated with vitronectin or laminin-1.
The vitronectin coating supported extensive network of
assembled fibronectin formed around cells expressing
wild-type 1A or D759A, Y783F, Y795F, or Y783,795F
mutant
1A (Fig. 5). In contrast, untransfected GD25 (Fig. 5) cells or cells expressing inactive T788P or
P781,793A mutant
1A (not shown) did not assemble fibronectin over this time period when cultured on vitronectin. Thus, assembly of a matrix by GD25 cells cultured
short-term on vitronectin requires active
1 integrins. Fibronectin deposition around cells expressing
1A or the
D759A mutation or the Phe for Tyr substitutions, whether cultured on vitronectin or laminin-1, consistently stained
at least as intensely as cells expressing wild-type
1A (Fig. 5).
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The qualitative fluorescence microscopic observations in
Fig. 5 were buttressed by assays of binding of 125I-labeled,
70-kD NH2-terminal fragment of fibronectin (Fig. 6). This
fragment is a suitable probe to quantify the ability of cells to assemble fibronectin, inasmuch as the fragment and intact fibronectin bind to cell layers with the same affinities,
cross-compete for binding, and can be cross-linked to the
same large apparent molecular mass target in cell layers
(46, 71). Cells expressing D759A or Y783,795F mutant
1A bound 1.5- to 2.5-fold more 70-kD fragment than
cells expressing wild-type
1A. Binding to cells expressing
1A with the single Tyr mutation was intermediate between binding to cells expressing
1A with the double Tyr
mutations and cells expressing wild-type
1A. Increased
binding was found regardless of whether cells were cultured on vitronectin, fibronectin, or laminin-1 and was enhanced by LPA, which causes a coordinated change in cell
shape, and upregulation of binding of fibronectin or the
70-kD fragment (45, 72).
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Cellular Localization of 1A in Relationship to
Assembled Fibronectin
Activated integrins are required in the process of fibronectin matrix assembly (69). We therefore analyzed cellular
localization of wild-type 1A or D759A or Y783,795F
mutant
1A in relationship to assembled fibronectin by
fluorescence microscopy of cells cultured on vitronectin,
fibronectin, or laminin-1 and incubated with FITC-fibronectin before fixation and staining for
1 (Fig. 7). These three
cell types in general behaved in the same manner on a
given substrate. However, they showed different subcellular localization of
1A that depended upon the different
substratum. In cells adherent to vitronectin,
1A did not
localize to focal contacts but rather to assembled fibronectin. In cells adherent to fibronectin, some
1A localized to
focal contacts of cells, and some was associated with assembled fibronectin, especially around cells expressing wild-type
1A and, to a lesser extent, around cells expressing D759A or Y783,795F mutant
1A.
1A localized to focal
contacts of all three cell types when cultured on laminin-1.
These focal contacts were linear and often associated with
FITC-fibronectin.
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Cell Motility and Migration of Cells Expressing
Wild-Type 1A or Mutant
1As
Motility of GD25 cells and the various transfectants was
assessed by an assay in which moving cells displace polystyrene beads layered onto surfaces coated with vitronectin, fibronectin, or laminin-1 (Fig. 8). GD25 cells cultured
in the presence of PDGF (Fig. 8) or EGF (not shown)
demonstrated little motility. GD25 cells expressing wild-type 1A or various mutants of
1A were not motile in
the absence of PDGF or EGF (not shown). Addition of
PDGF (Fig. 8) or EGF (not shown) caused greater motility of
1A-expressing cells, that is, a greater area around
cells was cleared of beads. The areas cleared by cells expressing wild-type
1A or the D759A mutant tended to be
asymmetrical and irregular whereas areas cleared by cells
expressing the Phe for Tyr substitutions, especially Y795F,
were rounder. These patterns were found regardless of
which adhesive protein (vitronectin, fibronectin, or laminin-1) was present on the surface and which motility agent
(EGF or PDGF) was present in the medium.
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Transwell migration assays were performed in blind-well chambers fitted with filters coated with vitronectin, fibronectin, or laminin-1. In the absence of EGF or PDGF, 4- to 10-fold more cells moved across filters coated with fibronectin (Fig. 9) than filters coated with gelatin (<10 cells/0.16 mm2; not shown). This increase is presumed to represent a haptotactic response. The haptotactic responses to vitronectin were both less in magnitude and less variable than the haptotactic response to fibronectin, whereas the haptotactic responses to laminin-1 were equally variable (Fig. 9). GD25 and Y783,795F cells did not demonstrate haptotaxis through laminin-1-coated filters.
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In response to EGF or PDGF, GD25 cells expressing
wild-type 1A or the D759A mutant migrated two- to
fourfold more than GD25 cells lacking
1A (Fig. 9). These
results indicate that migration is enhanced by
1A integrins interacting with the adhesive substrates coating the
filter, probably
v
1A with vitronectin,
6
1A with laminin-1, and
5
1A with fibronectin. In response to EGF,
D759A cells and cells expressing wild-type
1A migrated
equivalently. In response to PDGF, D759A cells migrated
better than cells expressing wild-type
1A. Y783,785F
cells, in contrast, migrated much less than cells expressing
wild-type
1A and no more than cells lacking
1A. Y783F
and Y795F cells had migratory behavior that was intermediate between Y783,795F cells and cells expressing wild-type
1A. In all experiments, however, migration of cells
expressing the Y783F mutation was less than migration of
cells expressing the Y795F mutation. Differences in migration ability among cells expressing various mutant
1As
were found over a 30-fold range of concentrations (3-100
ng/ml) of both of the chemotactic agents (not shown).
In the bead clearing assay, Y795F cells were most active,
and the Y783,795F cells were as active as cells expressing
wild-type 1A (Fig. 8). The discrepancy in behavior between the bead clearing assay and the chemotaxis assay
shown in Fig. 9 was investigated by checkerboard analysis
in which different concentrations of chemotactic agent are
added to the upper and lower chamber of the apparatus
(Table I). Such an analysis differentiates directed migration across the filter in response to a gradient of chemotactic agent (chemotaxis) from increased random motility because of the presence of the chemotactic agent per se
(chemokinesis). Cells expressing wild-type
1A and the
D759A mutant responded to a gradient of PDGF by
chemotaxis but demonstrated only a low and variable chemokinetic response to increasing concentrations of
PDGF in both chambers. The chemokinetic responses of
wild-type and D759A cells were more than twofold less
than the chemotactic responses. Migration of Y795F cells,
in contrast, was greater when PDGF was present in both
chambers than when there was a gradient of PDGF, indicating that the chemokinetic response was the main effect. Migration of Y783,795F and Y783F cells was low in magnitude and variable between fibronectin- and vitronectin-coated filters, making it difficult to classify the migration
as chemotaxis or chemokinesis.
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Focal Contact Formation and Actin Cytoskeleton
Organization in Cells Expressing Wild-Type 1A or
Mutant
1As
Cells expressing wild-type 1A and the D759A and
Y783,795F mutants were analyzed for focal contacts with
antipaxillin (Fig. 10 A) and antivinculin (Fig. 10 B) and for
F-actin-containing cytoskeleton with rhodamine phalloidin (Fig. 10 B). As assessed by paxillin or vinculin staining,
focal contacts formed by D759A cells on fibronectin were
larger, coarser, and more numerous than focal contacts
formed by cells expressing wild-type
1A. More numerous focal contacts were also formed by Y783,795F cells on fibronectin. Compared with the focal contacts formed by
D759A cells, focal contacts of Y783,795F cells were finer
and more uniform in size. The differences in the patterns
of paxillin and vinculin staining among the three cell types
on fibronectin were similar to the difference in staining
patterns of
1A (Fig. 7; more obvious in studies not shown
in which only single fluorescence for
1 was done). The
difference between vinculin-containing focal contacts of
D759A and Y783,795F cells was also found when the cells
were cultured on vitronectin-coated surfaces (Fig. 10 B)
even though
1A could not be demonstrated in focal contacts (see Fig. 7). On the vitronectin-coated substratum,
vinculin-staining focal contacts of cells expressing wild-type
1A were coarser and more heterogenous than wild-type cells on the other substrates. On a laminin-1-coated
substratum, more vinculin-staining focal contacts were
present in cells expressing wild-type
1A or the D759A
mutant than in Y783,795F cells.
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Cells expressing wild-type 1A or the D759A mutant,
when stained with rhodamine phalloidin, contained thick
cables that ran through most of the length of the cells (Fig.
10 B). These cables terminated in the coarse vinculin-staining focal contacts. The F-actin network of Y783,795F
cells was more peripheral than in cells expressing wild-type
1A or the D759A mutant and terminated in the fine
vinculin-staining focal contacts. The differences in F-actin
distribution were noted on all three substrata. For each of
the transfected cell types, however, the F-actin network was less when cells were on vitronectin. Each of the transfected cell types had a better developed F-actin network
than nontransfected GD25 cells on vitronectin or fibronectin (Fig. 10 B).
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Discussion |
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We studied eight cytoplasmic mutant of 1A
four active
(D759A, Y783F, Y795F, and Y783,795F) and four inactive
(P781A, T788P, P793A, and P781,793A) as judged by reactivity with the 9EG7 anti-
1 antibody, adhesion to laminin-1, and ability to support fibronectin assembly. (Recently, it was reported that human foreskin fibroblasts do
not assemble fibronectin when cultured on vitronectin
[18]. In our hands, human foreskin fibroblasts do assemble
fibronectin when cultured on vitronectin, and the inability
to assemble fibronectin when cultured on vitronectin is a
specific property of cells deficient in active
1 integrin
[Zhang, Q., T. Sakai, R. Fässler, J. Nowlen, and D.F.
Mosher, manuscript in preparation]). The active
1As were associated with distinctive cellular phenotypes.
D759A cells had thick F-actin-containing microfilaments
that terminated in coarse vinculin-containing focal contacts. Y783,795F cells had thinner and more peripheral
F-actin-containing microfilaments that terminated in fine
focal contacts. The cytoskeletal phenotypes were found
when cells were on vitronectin-coated substrates, in which
1A was associated with assembling fibronectin, as well as
on fibronectin- or laminin-1-coated substrates, in which
1A was associated both with focal contacts and assembling fibronectin. Motility phenotypes were associated with the
cytoskeletal phenotypes. D759A cells were as active as
cells expressing wild-type
1A in chemotaxis assays whereas
Y783,795F cells were no more active than cells lacking
1A and Y795F demonstrated a strong chemokinetic response.
Cell migration is a complex process that involves lamellipodial extension, generation of intracellular force, integrin clustering in focal contacts, integrin avidity, and integrin signaling (34, 39, 51, 61). Focal contacts are enriched
in a large number of kinases, at least one phosphatase, and
various adapter and connector proteins (11, 22, 27, 38, 44,
70). Phe lacks the hydroxyl group that is the acceptor
site for tyrosine phosphorylation. The results suggest,
therefore, that conversion of tyrosines between nonphosphorylated and phosphorylated states is critical for movement on adhesive substrates (Fig. 11). Phosphorylated integrins are hypothesized to initiate a pathway leading to
changes in F-actin-containing cytoskeleton and generation of the polarity required for directional movement.
NPXY motifs are also hypothesized to regulate cellular localization of 1A integrins. Cells lacking focal adhesion tyrosine kinase (FAK) exhibit reduced motility and increased numbers of focal contacts whereas overexpression
of FAK causes increased motility (14, 36). Enhancement
of tyrosine phosphorylation causes loss of focal contacts
(20, 47). Phosphorylated
1 integrins of src-transformed
fibroblasts do not localize to focal contacts (37). Solubilized phosphorylated integrins from src-transformed fibroblasts, in contrast to integrins from normal fibroblasts, fail
to bind to an extracellular ligand, fibronectin, or cytoplasmic protein, talin (63). These observations all suggest that
upon phosphorylation of the NPXY motifs by tyrosine kinase(s), the integrin loses its affinity for both extracellular
ligand and cytoplasmic components of the focal contacts
and exits the focal contact. Dephosphorylation of the motifs by unknown tyrosine phosphatase(s) would allow the
integrin to participate in a new round of ligation and focal
contact formation. The two hypotheses shown in Fig. 11
could be linked in the sense that the cytoskeleton may polarize the cycling of integrins.
|
For phosphorylated integrins to initiate a pathway leading to changes in F-actin-containing cytoskeleton, one or
more adaptor molecules specific for the phosphorylated
NPXY motifs would be required. Tyrosine-phosphorylated intracellular domains interact with Grb2 and Shc in
the case of 3 (40) and with the P85 of phosphoinositide-3
kinase (PI-3) kinase in the case of
1A (37). However, the
double Phe for Tyr substitution did not impair the ability
of human
1A transfected into mouse 3T3 cells to signal via Shc when cross-linked by mouse anti-human
1 antibody (66). D759A and Y783,795F cells each had increased
numbers of focal contacts and upregulation of fibronectin
assembly. Increased focal contacts also correlate with increased assembly when cells are treated with LPA (2, 45,
55, 72, 73). The patterns of focal contacts in D759A and
Y783,795F cells, however, were strikingly different. The
different patterns and the divergent behavior of the two
cells in the migration assay suggest that the mutations influence focal contacts by impinging at different points in
the pathway shown in Fig. 11. The D759A mutation likely increases the avidity of
1A integrins for extracellular
ligands and the probability that integrins will bind ligand
and enter focal contacts (32). Increased avidity of integrins
for ligands does not slow migration but rather lowers the
density of ligands required for maximum migration (12,
34, 39, 51). The Y783,795F mutations, as above, are hypothesized to cause persistence of ligated
1A integrins in
focal contacts.
3 in which the tyrosine homologous to Y783 in
1A
was mutated to phenylalanine failed to induce adhesion
and clot retraction by cells in which
3 integrins are constitutively inactive (9). Our tyrosine mutants of
1A were
constitutively active in GD25 cells. We do not know
whether expression of the mutant
1As in cells in which
the integrins are constitutively inactive would reveal additional functions for the phosphorylation. Indeed, the argument that phosphorylation rather than the simple absence
of the hydroxyl group explains the observed phenotype of
transfected GD25 cells is circumstantial. The argument is
based on the fact that
1A can, in principle, be phosphorylated as evidenced by studies of transformed cells and presumes that only a small, undetectable fraction of
1A is
phosphorylated at any one time in untransformed cells.
Several of the substitutions generated in the present investigation are found naturally in other subunits (Fig. 1).
In
1D, the conserved Thr in the sequence between the
NPXY motifs is an Asn, and a Pro is present in the sequence.
1D is a muscle-specific integrin found in junctions where there is high tensile strength (5).
1D-transfected
GD25 display retarded spreading, reduced cell migration,
enhanced contractility, and robust ability to assemble fibronectin matrix when compared with
1A-expressing counterparts (6). Transfected
1D is targeted to focal contacts and is also more strongly associated with detergent-insoluble cytoskeleton than
1A. These results suggest
that alternative splicing of
1 is a means to strengthen the
cytoskeleton-matrix link where an extremely stable association is required for contraction (6). In
2, Tyr of both
NPXY motifs are replaced by Phe.
2 integrins interact
with counter receptors on other cells, thus localizing lymphocytes and leukocytes in inflammatory and immunological reactions and facilitating interactions among these
cells (13, 26, 30, 35, 56). These and other (10, 24, 30, 35, 52,
53, 64, 68) natural variations in NPXY sequences in
subunits may determine the kinetics with which integrins enter and exit focal contacts and the relative abilities of integrins to mediate cell migration, stable adhesions, endocytosis,
and assembly of extracellular matrix.
![]() |
Footnotes |
---|
Received for publication 1 December 1997 and in revised form 26 February 1998.
Address all correspondence to Deane F. Mosher, Departments of Medicine and Biomolecular Chemistry, University of Wisconsin-Madison, 1300 University Avenue, Madison, WI 53706. Tel.: (608) 262-1576. Fax: (608) 263-4969.We gratefully acknowledge J. Nowlen, K. Schell, and C. Dizack for valuable technical help, W. Busse for his interest and encouragement, S. Miyamoto for suggestions regarding cDNA constructs, and M. Salmon for preparation of the manuscript.
This work was supported by National Institutes of Health grants HL21644 and HL54462, fellowship funds from the Cell Science Research Foundation and Marion Merrill Dow (to T. Sakai), and a grant from the Swedish Medical Research Foundation.
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Abbreviations used in this paper |
---|
FAK, focal adhesion kinase; LPA, lysophosphatidic acid; LRSC, lissamine rhodamine B sulfonyl chloride.
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