(Received for publication, September 13, 1995; and in revised form, November 30, 1995)
From the
Distinctions between chemotaxis and haptotaxis of cells to
extracellular matrix proteins have not been defined in terms of
mechanisms or signaling pathways. Migration of A2058 human melanoma
cells to soluble (chemotaxis) and substratum-bound (haptotaxis)
vitronectin, mediated by , provided a
system with which to address these questions. Both chemotaxis and
haptotaxis were completely inhibited by treatment with RGD-containing
peptides. Chemotaxis was abolished by a blocking antibody to
(LM609), whereas haptotaxis was
inhibited only by approximately 50%, suggesting involvement of multiple
receptors and/or signaling pathways. However, blocking antibodies to
, also present on A2058 cells, did
not inhibit. Pertussis toxin treatment of cells inhibited chemotaxis by
>80%, but did not inhibit haptotaxis. Adhesion and spreading over
vitronectin induced the phosphorylation of paxillin on tyrosine. In
cells migrating over substratum-bound vitronectin, tyrosine
phosphorylation of paxillin increased 5-fold between 45 min and 5 h.
Dilutions of anti-
that inhibited
haptotaxis also inhibited phosphorylation of paxillin (by
50%) and
modestly reduced cell spreading. In contrast, soluble vitronectin
(50-100 µg/ml) did not induce tyrosine phosphorylation of
paxillin. The data suggest that soluble vitronectin stimulates
chemotaxis predominantly through a G protein-mediated pathway that is
functionally linked to
. Haptotaxis
is analogous to directional cell spreading and requires
-mediated tyrosine phosphorylation of
paxillin.
Active locomotion of tumor cells stimulated by cytokines and ECM ()proteins contributes to invasion and metastasis of
malignant neoplasms(1, 2, 3) . Migration
induced by ECM proteins is further defined as chemotaxis (CTX) when the
ligand is soluble and haptotaxis (HTX) when substratum-bound. Tumor
cells traversing blood vessels and tissue stroma interact with intact,
substratum-bound as well as soluble, partially degraded ECM components (4, 5) ; in addition, fibronectin and vitronectin are
present in plasma (6, 7, 8) . Therefore,
chemotactic and haptotactic migration of tumor cells has physiological
relevance and involves binding of cell surface integrins to their
respective ligands. Much is being learned about integrin-mediated
signaling and the intracellular signals underlying cell
migration(4, 7, 9, 10) . However,
mechanistic differences between CTX and HTX that may be secondary to
differences in signaling have not been described.
Previously, the
human melanoma cell line A2058 was shown to migrate directionally in
response to soluble and substratum-bound laminin, type IV collagen, and
fibronectin(3) . CTX and HTX to collagen IV were distinguished
by PT sensitivity, although the receptor(s) involved were not
identified. Migration of these cells to vitronectin, another adhesive
glycoprotein that can influence invasion and metastasis, had not been
studied. VN is found in plasma and transiently in the ECM, where it has
regulatory roles in coagulation and pericellular proteolysis,
respectively(6, 8, 11) . Several studies have
reported a potential involvement of VN and the VN receptor
in the pathogenesis and progression
of malignant
melanoma(12, 13, 14, 15) . In
vitro, VN mediates attachment and spreading of cultured cells
through interaction of its amino-terminal Arg-Gly-Asp (RGD) domain with
cell surface
integrins(13, 16, 17, 18) . Of
the known integrin receptors for VN, including
,
,
, and
(7) ,
is most frequently associated with
cell spreading and migration over VN-coated
substrates(6, 17, 19, 20, 21, 22, 23) .
In the present report, CTX and HTX of A2058 cells to VN was found to be
mediated by a common receptor,
. This
system allowed study of the distinct signaling mechanisms involved in
these two types of migration.
Results are expressed as stimulated motility, which represents the total motility response minus unstimulated random motility.
In one set of experiments (Fig. 7), cells were set up in four large single-well chambers
(volume of lower wells is 12 ml, volume of upper wells is 9 ml, and
they accommodate 25 80-mm filters). Triton X-100 lysates of
migrating cells were collected at various time points for SDS-PAGE and
immunoblotting. At 45 min, when cells had just attached to the upper
filter surface, one chamber was disassembled, and all cells on the
filter were collected in lysis buffer (see below). At all subsequent
time points (90 min, 3 h, and 5 h), cells on the upper filter surface
were first removed, allowing the separation and collection of only the
migrating cells for lysis.
Figure 7: Paxillin is phosphorylated on tyrosine as cells migrate over substratum-bound VN. HTX was performed in four parallel single-well chambers. At 45 min (lane 1), 90 min (lane 2), 3 h (lane 3), and 5 h (lane 4), chambers were disassembled and Triton X-100 lysates of migrating cells were collected as described under ``Materials and Methods.'' 11 µg/lane of lysate was immunoblotted with anti-phosphotyrosine. Integrated densities of phosphotyrosine bands, normalized as described under ``Results,'' are as follows: lane 1, 0.35; lane 2, 0.79; lane 3, 1.22; lane 4, 1.43. This experiment was repeated twice, with similar results.
For Western immunoblotting, samples were electrophoresed under reducing conditions according to the method of Laemmli (26) through 8-16% precast gradient gels. Proteins were then electroblotted onto polyvinylidene difluoride membranes (Novex), and membranes were blocked in casein-containing buffer before incubation with antibodies. Blots were developed using the ECL chemiluminescence detection kit (Amersham Corp.)/
Blots were scanned with an Agfa photoscanner, using Adobe Photoshop 3.0 to create images for figures. Bands were quantitated by densitometry using Image 1.49. Blots chosen for scanning and quantitation were only those exposures that gave chemiluminescence signals within the linear range of detection.
Figure 1:
Soluble VN stimulated CTX of A2058
human melanoma cells, which peaked at 100 µg/ml VN to a value
4-fold above background migration (subtracted out in all figures).
Graph illustrates one experiment, representative of
four.
We also observed a strong haptotactic response of A2058 cells to substratum-bound VN, maximal at a coating concentration of 10 µg/ml (data not shown).
Adhesion of -containing integrins
to VN is mediated by the RGD sequence at the amino terminus of
VN(8, 27) . To assess the importance of receptor
binding to this sequence in VN-mediated motility, cells were pretreated
with a pentapeptide containing the RGD sequence (GRGDS) at 500
µM prior to CTX and HTX assays. The RGD-containing peptide
abolished both CTX and HTX to VN (data not shown), indicating that
integrin binding to the RGD site is an absolute requirement for both
types of motility. Pretreatment with the control peptide GRGES at the
same concentration resulted in 20-40% inhibition.
Pertussis
toxin ADP-ribosylates the subunit of certain classes of
heterotrimeric GTP-binding proteins. This results in the functional
uncoupling of G proteins from their receptors, blocking signal
transduction(28) . Since CTX of A2058 cells to laminin and type
IV collagen was inhibited by treatment of cells with PT(3) , we
tested the effects of PT on CTX and HTX to VN (Fig. 2). Cells
were treated with PT at the indicated concentrations, then tested for
motility to soluble VN (50 µg/ml) (A) and substratum-bound
VN (B). As illustrated, PT treatment results in a
concentration-dependent inhibition of CTX, with maximal inhibition
(
100%) at 0.5 µg/ml (Fig. 2A). This
concentration of PT also completely inhibited CTX to higher
concentrations of VN (100-400 µg/ml; data not shown).
However, the same batch of cells migrated at or near control levels
over substratum-bound VN at all PT concentrations (Fig. 2B). These results implicate a PT-sensitive G
protein in transduction of the chemotactic signal through
, whereas this signal transduction
pathway does not regulate VN-mediated HTX. PT at 0.5 µg/ml did not
inhibit adhesion or spreading of melanoma cells on VN- or
gelatin-coated dishes (data not shown). Treatment with this
concentration of PT is sufficient to completely ribosylate G proteins
in these cells (data not shown).
Figure 2: Pertussis toxin treatment results in concentration-dependent inhibition of CTX, but not HTX, to VN. Cells were pretreated with PT at the indicated concentrations, then assayed for motility to soluble (50 µg/ml) (A) or substratum-bound (B) VN. Graph illustrates one experiment, representative of three.
The VN receptor was also
identified by immunoprecipitation from
[
S]methionine-labeled lysates with monoclonal Ab
LM609 (Fig. 3). SDS-PAGE under nonreducing conditions followed
by autoradiography revealed bands of approximately 160 kDa (
chain) and 95 kDa (
chain) in the
immunoprecipitate (lane 4). Under reducing conditions (lane 2), the
chain migrates at
140 kDa
and the
chain at
110 kDa. Integrin
was not detectable by
immunoprecipitation with anti-
(P1F6) (lanes 1 and 3).
Figure 3:
Immunoprecipitation of
from A2058 cells. Cells were
pulse-labeled with
S-methionine, after which Nonidet
P-40-extracted lysates were immunoprecipitated with
anti-
(lanes 2 and 4). Under nonreducing conditions (lane 4), a
heterodimer of
160 kDa (
chain) and
95 kDa (
chain)
was detected. Under reducing conditions (lane 2), the
chain migrated at
140 kDa and the
chain at
110 kDa.
Integrin
was not detected by
immunoprecipitation under the same conditions using
anti-
(lanes 1 and 3).
Immunofluorescent staining
revealed the differential distribution of and
on attached and spread
A2058 cells (data not shown).
is
localized to focal adhesions, which were predominant along the cell
margins, whereas
had a more diffuse,
nonfocal contact distribution throughout the cell, similar to previous
observations in other human tumor cell lines(19) .
Figure 4:
is the
primary CTX receptor and is a major receptor for HTX to VN. A,
anti-
inhibits CTX to VN in a
concentration-dependent manner. Cells were treated with
anti-
(LM 609) at the indicated
dilutions, then assayed for CTX to 50 µg/ml VN in the continued
presence of antibody. Maximal inhibition of stimulated motility
(
90%) was observed at 1:10,000. Graph illustrates one experiment,
representative of three. B, anti-
inhibits HTX. Cells were treated with antibodies to
,
,
or the
subunit at 1:1000. One aliquot of cells was
given a combination of anti-
plus
anti-
, each at 1:1000. Cells were then assayed for
motility to substratum-bound VN in the continued presence of antibody.
Anti-
inhibited HTX by
49%,
relative to untreated cells. Graph illustrates one experiment,
representative of four.
Whereas
anti- treatment of A2058 cells nearly
abolished CTX to VN, the same dilutions of Ab did not inhibit CTX to
type IV collagen or laminin (data not shown), confirming the
involvement of distinct receptors in CTX to these matrix components.
A2058 cells also express on their
surfaces, although at lower levels. This integrin mediates migration of
keratinocytes (29) and pancreatic carcinoma cells (30) over VN-coated substrata. As expected, however, treatment
of A2058 cells with blocking Abs to
(P1F6) or to the
subunit (JB1a) did not inhibit
stimulated CTX to VN (at 1:200-1:10,000) (data not shown).
As
with CTX, HTX to VN was inhibited by treatment of cells with
anti- (Fig. 4B), and
this inhibition was maximal at an antibody dilution of 1:10,000 (data
not shown). Unlike CTX, inhibition of HTX by
anti-
ranged from only 40% to 60% in
several experiments. To test the possibility that
and/or a
integrin
contributed to HTX on VN, cells were treated (at 1:1000) with the
corresponding blocking Abs and assayed for migration (Fig. 4B); one aliquot of cells was treated with a
combination of Abs to
and
, each at 1:1000, to determine if additive inhibition
would be observed. However, only anti-
significantly inhibited HTX to VN (49%). Cells treated with
anti-
plus anti-
were not inhibited to any additional degree relative to those
treated with anti-
alone. A range of
dilutions of anti-
and isotypic
control antibody had no inhibitory effect on HTX (data not shown).
Therefore, although anti-
did not
inhibit HTX as completely as it inhibited CTX,
still appears to be the major
motility-promoting receptor in HTX; the other VN receptors
and
do not play a direct role.
Figure 5: Substratum-bound VN induces tyrosine phosphorylation of paxillin. A, cells were plated on VN-coated (lane 1), gelatin-coated (lane 2), or tissue culture plates (lane 3) and allowed to adhere for 90 min. Cells were lysed in Triton X-100, and 10 µg of each lysate was electrophoresed and immunoblotted with anti-phosphotyrosine (PY20). Note the 68-kDa phosphotyrosine in lysates of VN-plated cells (lane 1), which is absent from the other lanes. B, paxillin was immunoprecipitated with anti-paxillin mAb from 120 µg of the lysate used in panel A (lane 1). Immunoprecipitate was divided into two portions for duplicate immunoblots with anti-phosphotyrosine (anti-PY, lanes 1-3) and anti-paxillin (anti-Pax, lanes 4-6). Lanes 1 and 4, anti-paxillin immunoprecipitate; lanes 2 and 5, 10 µg of non-immunoprecipitated lysate; lanes 3 and 6, 120 µg of lysate immunoprecipitated with isotype control Ab (mouse IgG1). The 68-kDa phosphotyrosine in lane 2 co-migrates with immunoprecipitated paxillin (lane 1, arrow). (Lower bands in lanes 1 and 4 are probably antibody chains in the immunoprecipitates.)
Fig. 5A illustrates that adhesion is sufficient to induce tyrosine phosphorylation of the 110-130-kDa cluster, but the 68-kDa band is detectably phosphorylated on tyrosine only in VN-adherent cells (lane 1). We suspected the 68-kDa band to be paxillin, based on its molecular mass and phosphorylation on tyrosine upon adhesion (31) . Paxillin is phosphorylated on tyrosine in response to a variety of signals, all involving cytoskeletal remodeling(32) . From the lysate of VN-adherent cells (Fig. 5A, lane 1), we immunoprecipitated paxillin with monoclonal Ab to paxillin. In Fig. 5B, the immunoprecipitate was divided into two portions for Western immunoblotting with anti-phosphotyrosine (lanes 1-3) and anti-paxillin (lanes 4-6). In lane 1, the anti-paxillin immunoprecipitate stains prominently at 68 kDa (arrow) with anti-phosphotyrosine. In lane 2, non-immunoprecipitated lysate was run alongside for comparison. Lane 4 is immunoprecipitated paxillin, which stains with anti-paxillin; this band co-migrates with a band from the nonfractionated lysate (lane 5). (Lanes 3 and 6 are immunoprecipitates of this lysate using isotype control mAb.) These results show that A2058 cells do contain paxillin, and that paxillin is a protein phosphotyrosine of 68 kDa in VN-adherent cells.
Tyrosine phosphorylation of paxillin increased over time in
VN-adherent cells, coincident with cell spreading (Fig. 6). To
determine the time course of phosphorylation, cells were plated on
VN-coated dishes, and lysates were collected after 10 min (lane
3), 20 min (lane 4), 45 min (lane 5), 90 min (lane 6), and 4 h (lane 7); for comparison, two
aliquots of cells were left in suspension for 90 min (lane 1,
room temperature; lane 2, 37 °C). Fig. 6A shows the anti-phosphotyrosine immunoblot (68-kDa region) of
lysates from each time point (10 µg/lane). In Fig. 6B, a duplicate blot probed with anti-paxillin
illustrates that nearly equal amounts of total paxillin were loaded per
lane. To quantitate and correct for slight under- or overloading of
lanes relative to lane 1 (using the paxillin signal in panel B as a standard), bands were quantitated by densitometry
as described under ``Materials and Methods.'' For each lane
we obtained a ratio of anti-phosphotyrosine to anti-paxillin signals;
in lanes 2-7, these ratios were maintained as each
paxillin value was adjusted to that of lane 1, and each
phosphotyrosine value was correspondingly adjusted. In this manner, a
more accurate comparison of phosphotyrosine signals between lanes is
obtained. Fig. 6C shows the normalized values for
phosphotyrosine signals (PY20); numbering of lanes corresponds to Fig. 6(A and B). After 10 min on VN, cells
were adherent, but remained round (photomicrograph, panel A);
these lysates had no detectable phosphorylated paxillin (lane
3, Fig. 6, A and C). However, with
increasing time of adhesion and spreading on VN (photomicrograph, panels B-D), tyrosine phosphorylation of paxillin
increased 7-fold until it reached a maximum at 90 min (lane
6, Fig. 6, A and C), coincident with
maximal spreading (photomicrograph, panel D). At 4 h (lane
7, Fig. 6, A and C), phosphorylation
remained at peak levels or declined somewhat.
Figure 6:
Tyrosine phosphorylation of paxillin
gradually increases in cells adherent to VN. Cells were plated on
VN-coated dishes; at the indicated times after plating, lysates were
collected as described under ``Materials and Methods.'' A, 10 µg/lane of each lysate was immunoblotted with PY20
(68-kDa region illustrated). B, duplicate immunoblot was
stained with anti-paxillin. Lanes 1 and 2, cells in
suspension for 90 min at room temperature (lane 1) and 37
°C (lane 2); lanes 3-7, cells adherent to
VN for 10 min (lane 3), 20 min (lane 4), 45 min (lane 5), 90 min (lane 6), and 4 h (lane 7). C, table showing the integrated densities of phosphotyrosine
bands (panel A), normalized as described under
``Results.'' Lanes are numbered as in panels A and B. This experiment was repeated three times, with similar
results. Lower panels, cells were fully spread on VN-coated
dishes within 90 min. At 10 min (A), 20 min (B), 45
min (C), and 90 min (D) after plating, adherent cells
were processed and photographed with a Zeiss Axiophot microscope under
phase contrast. Magnification, 100.
In contrast, soluble VN at concentrations that stimulate CTX (50-100 µg/ml) did not induce detectable tyrosine phosphorylation of paxillin in gelatin-adherent cells (data not shown), when examined over a time course of 5 min to 3 h. We also did not detect tyrosine phosphorylation of paxillin in cells migrating to gradients of soluble VN, in experiments using single-well chambers as in Fig. 7(data not shown).
Figure 8:
Anti-
treatment of A2058 cells results in reduced tyrosine phosphorylation of
paxillin (arrow in panel A) and modestly reduces cell
spreading. Cells were treated with anti-
or anti-
(1:1000), then plated
onto VN-coated dishes, and allowed to adhere for 90 min. Lysates were
prepared as described, and 10 µg/lane of lysate was immunoblotted
with anti-phosphotyrosine (A) or duplicate blot probed with
anti-paxillin (B). Lane 1, untreated cells; lane
2, anti-
; lane 3,
anti-
. C, table showing
integrated densities of the 68-kDa phosphotyrosine bands (panel
A), normalized as described under ``Results.'' This
experiment was repeated twice, with similar results. Photomicrographs
on right: cells were left untreated (A) or were treated with
(1:1000) mouse IgG
(B),
anti-
(C), or
anti-
(D); after adhesion to
VN for 90 min, adherent cells were processed and photographed as in Fig. 6. Magnification,
100.
Our data demonstrate that A2058 cells have a high ratio of
to
integrins on their surfaces and migrate rapidly toward
concentration gradients of soluble and substratum-bound VN. While both
CTX and HTX are mediated primarily by interaction of
with the RGD sequence in VN, there
are differences in signal transduction mechanisms and phosphorylation
patterns elicited by soluble versus substratum-bound VN, which
may differentially affect the motility machinery. Specifically, CTX to
VN is inhibited 80-100% by pertussis toxin, implicating a
heterotrimeric G
-like protein in signal transduction. In
contrast, transduction of signals for HTX is largely independent of
such a G protein. Blocking antibodies to
completely inhibited VN-stimulated CTX, while HTX is inhibited by
only
50%. Finally, the focal adhesion protein paxillin becomes
tyrosine phosphorylated upon melanoma cell spreading and migration over
substratum-bound VN in an
-dependent
manner, whereas soluble VN (at concentrations stimulating CTX) does not
induce detectable tyrosine phosphorylation of paxillin. These
differences may reflect quantitative and qualitative differences in the
signals that regulate the motility apparatus.
Studies of agonist-stimulated eukaryotic cell migration have not revealed a unifying intracellular signaling pathway, despite overall similarities in crawling mechanisms(33) . Even within the same cell type, different attractants can induce motility through distinct signaling pathways(2, 17, 25, 34) . Previous studies with A2058 cells distinguished CTX from HTX by signal transduction through a PT-sensitive G protein(3) . The receptor(s) involved were not identified; however, these studies showed that the same ECM protein could stimulate motility through distinct pathways when soluble versus substratum-bound. There has remained uncertainty as to whether CTX and HTX are truly distinguishable; other studies have not defined differences between them(23) . Study of VN-mediated migration of A2058 cells allowed further investigation of the following questions. What additional signals distinguish CTX from HTX? How could the soluble and substratum-bound forms of an ECM protein stimulate different signaling pathways in the same cell? Would this result in distinct mechanisms of migration?
ECM proteins induce intracellular signals in large part
through integrin receptors(4, 7) . In this report,
identification of integrin as the
motility-promoting receptor (particularly for CTX) through the use of
blocking antibodies allowed additional insights into these questions.
Ligand binding to
in other systems
induces elevation of intracellular Ca
(17) ,
protein tyrosine phosphorylation (35, 36, 37) , and collagenase
secretion(15) . Many integrin-generated signals require
clustering of integrins by adhesion to ECM, or with anti-integrin
antibodies(38, 39, 40) , as opposed to simple
occupancy by monovalent, soluble ligand. However, Miyamoto et
al.(39) identified distinct and increasing effects of
integrin occupancy, aggregation, and both combined. This illustrates
the diverse and graded nature of integrin-generated signals, and the
importance of the form in which ligand is presented. Extrapolating to
our studies, soluble and substratum-bound VN may cluster and activate
to different degrees, generating
distinct signals. Differential signaling could also be due to different
fates of the ligand-receptor complexes. Integrins cross-linked by
soluble antibodies are rapidly internalized, leading to termination of
signals(38, 40, 41, 42) . If soluble
VN is internalized more rapidly than substratum-bound
VN(43, 44) , chemotactic signals may not be as strong
or as sustained as those for HTX.
Although HTX to VN is mediated by
, additional receptors of varying
affinities and specificities may also contribute, resulting in multiple
motility signals that could act synergistically. CTX, in which the
soluble attractant can diffuse in three dimensions, would favor only
the highest affinity ligand-receptor interaction, and therefore one
predominant signaling pathway would be expected. HTX, in which the
attractant is immobilized in two dimensions, would be more permissive
for multiple low affinity interactions of VN with the cell surface.
This would explain the inability of one agent to completely inhibit
HTX. HTX was not inhibited by blocking Abs to
or to the
subunit,
or with exogenous heparin (data not shown). This indicates that binding
of these integrins and cell surface proteoglycans to VN does not
directly induce HTX; however, it does not rule out secondary,
modulatory roles. An example of cooperativity between integrins was
reported (20) in which the ``collaborating'' integrin
did not bind to the substrate.
PT sensitivity has been noted for a
number of chemotactic signaling pathways, notably in neutrophils (45) and melanoma cells, mentioned above. Heterotrimeric G
proteins transduce chemotactic signals in neutrophils in part by
activation of phosphatidylinositol metabolism (9, 45, 46, 47, 48) . The
products of these pathways are then thought to mediate actin
polymerization, cross-linking, and pseudopod
protrusion(33, 46, 49, 50) . The
mechanism by which integrin couples
to a G protein in VN-mediated CTX is not known. However, several
reports describe systems in which heterotrimeric G proteins are
functionally linked to ``non-classic'' receptors including
cell adhesion molecules(51, 52) , epidermal growth
factor receptor(53, 54) , and
others(55, 56, 57, 58) . Additional
studies will address the identity of the G
-like protein
mediating CTX to VN, the nature of its coupling to
, and downstream effectors.
Tyrosine phosphorylation of paxillin, a 68-kDa focal adhesion
protein (32) , accompanies cell spreading and HTX on VN. Upon
anti- treatment, tyrosine
phosphorylation of paxillin and cell spreading are both reduced by
50%, observed after 90 min on VN. The corresponding inhibition of
HTX is observed at a later time (5 h), indicating that paxillin
phosphorylation and cell spreading are early events that are necessary
for HTX. Signal transduction through integrins and other receptors to
the cytoskeleton is mediated by phosphorylation of paxillin, resulting
in cytoskeletal
remodeling(31, 32, 59, 60, 61, 62, 63, 64) .
Its multiple tyrosine phosphorylation sites and its potential
interactions with signaling molecules and cytoskeletal proteins in
focal adhesions(10, 32, 60) make paxillin a
likely candidate for a key regulator of cell spreading and HTX.
In
conclusion, the data suggest that different mechanisms of migration
predominate in CTX versus HTX. HTX involves an initial
interaction of with the RGD sequence
of substratum-bound VN, clustering of
, and phosphorylation of paxillin.
Additional signals generated by
ligation (and perhaps other receptors) promote actin
polymerization and linkage of filament bundles with
integrins(65) . The gradient of VN favors formation of adhesive
contacts and polymerized actin at the ``leading edge'' of
cells. HTX could therefore be described as continuous, directional
spreading, with the VN gradient maintaining directional cues.
Detachment of the trailing edge to allow forward locomotion may require
a Ca
-activated phosphatase(34) . In CTX,
adheres to gelatin-coated filters
for traction, but the gradient of soluble VN provides a directional
motility cue. The predominant signaling pathway is G protein-mediated;
this may involve localized phosphatidylinositol metabolism and cycles
of pseusopod protrusion and cell translocation in a manner analogous to
that described for neutrophils and Dictyostelium(9, 33) . Apparently the
tyrosine phosphorylation of paxillin is not involved in CTX. Our
preparation of VN is purified using urea and, therefore, is
multimeric(8, 66, 67) . Soluble, multimeric
VN at high concentrations (400-600 µg/ml) clusters integrin
and stimulates tyrosine
phosphorylation in endothelial cells(36) . In our system,
however, soluble VN at optimal concentrations for CTX did not stimulate
detectable tyrosine phosphorylation. The versatility of integrins as
signaling receptors has implications for a wide range of biological
processes, including invasive tumor cell migration.