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* This work was supported by the Swedish Research Council, the Swedish Cancer Foundation, the King Gustavs Vth 80 year Foundation, the Medical Faculty Research Foundation at Umeå University and the Infection and Vaccinology Research Program of the Swedish Foundation for Strategic Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The on-line version of this article (available at http://www.jbc.org) contains four additional figures. ‡ Recipient of stipends from the J. C. Kempe Memorial Foundation and from the Wallenberg foundation.
Invasin-promoted spreading of β1-integrin-deficient cells, transfected with the β1A- or β1B-integrin splice variants, were used to dissect early β1-integrin signaling events. The β1B isoform, which has a different membrane-distal part of the cytoplasmic tail from β1A, is defective in signaling and function. When plated on surfaces coated with the high affinity ligand invasin, β1B-integrin-expressing cells spread by forming filopodia with distinct adhesive phosphotyrosine complexes at the tips, without signs of lamellipodia. This suggested that the β1B-integrin mediated a partial signaling sufficient for formation of filopodia but insufficient for lamellipodia formation. When screening for proteins present in the distal filopodial phosphotyrosine complexes of β1B cells, p130Cas and the filopodia proteins vasodilator-stimulated phosphoprotein and talin were found, whereas the typical focal complex proteins focal adhesion kinase, paxillin, and vinculin were not. Invasin-promoted adhesion induced complex formation of p130Cas and the adapter Crk. Moreover, Crk together with Dock180 were present at the filopodial tips of β1B-integrin-expressing cells, and there was a prominent Rac1 activation. Expression of dominant negative variants of p130Cas or CrkII blocked β1B-integrin-mediated filopodia formation, indicating that this signaling scaffold is central in this process.
Integrins are a large family of at least 24 heterodimeric transmembrane receptors formed by 18 α and 8 β subunits (
). They are involved in bidirectional signaling across the cell membrane and influence several cellular events including cell spreading, cell migration, and cell survival (
). Integrin receptors link the extracellular matrix to the cytoskeleton at sites known as cell-matrix adhesions, which are multimolecular structures where the extracellular matrix, integrins, filamentous actin (F-actin),
). Cells form different types of cell-matrix adhesions that are morphologically distinct and are divided into subtypes such as focal contacts/focal adhesions and focal complexes. Focal contacts represent the relatively large and arrow-shaped cell-matrix adhesion structures mainly found at the basolateral plane of the cell, whereas focal complexes represent the small round-shaped cell-matrix adhesions usually present at the rim of lamellipodia (
). One protein family that regulates the cell-matrix adhesions and the actin cytoskeleton during cell spreading and migration is the Rho family of GTPases that includes Rho, Rac, and Cdc42 (
). During stimulated migration of NIH3T3 cells, Cdc42 is activated and induces formation of thin protrusions rich in actin, known as microspikes or filopodia (
). RhoA is also activated and mediates formation of stress fibers and focal contacts that firmly anchor the cell to the substratum and allow retraction of the rear end of the cell (
Among the proteins found in cell-matrix adhesions are FAK, Src, Crk-associated substrate (p130Cas), talin, vasodilator-stimulated phosphoprotein (VASP), paxillin, and vinculin (
). Vinculin plays a central role in the mechanical coupling of integrins to the cytoskeleton as well as in related control of cytoskeletal mechanics, cell shape, and motility (
). VASP, which also is found in the tip of filopodia, is involved in regulation of actin-based types of cell movement and promotes the formation of long actin filaments over branching (
). Paxillin can interact with a variety of signaling proteins and is presumed to be involved in several signaling pathways as well as in anchoring of the actin cytoskeleton (
). FAK is a tyrosine kinase that is activated early in response to integrin stimulation and is involved in regulating the turnover of cell-matrix adhesions (
). This kinase, together with members of the Src family of cytosolic tyrosine kinases, binds and phosphorylates the docking proteins p130Cas and paxillin (
). Phosphorylated p130Cas can interact with Crk, which in turn binds Dock180, which is an unconventional Rac-guanine nucleotide exchange factor that activates Rac1 (
), was unable to bind to a β1-integrin-deficient cell line, GD25, unless this cell line had been transfected with either of two splice variants of β1-integrin, β1A or β1B. These splice variants are identical in the extracellular, transmembrane, and membrane-proximal 20 amino acids of the cytoplasmic tail but differ at the end of the cytoplasmic tail (
). The distal part of the β1A tail contains 21 amino acids encompassing two NPXY motifs, a serine and a double threonine phosphorylation site. The unique part of the β1B tail does not contain any known motifs important for the function of β1B when it localizes to the cell surface although there is a double lysine motif that can cause intracellular accumulation of β1B (
). The β1A splice variant is most commonly expressed and can cluster, form cell-matrix adhesions, and mediate signaling upon binding to fibronectin or laminin. The β1B splice variant, on the other hand, is only expressed in a few human cell types and is unable to bind fibronectin unless stimulated with manganese ions that force the integrin into an active conformation, but even when binding an extracellular matrix ligand, the β1B variant fails to form detectable cell-matrix adhesions or induce signaling through FAK (
). We previously showed that GD25 cells complemented with β1A (GD25β1A) bound invasin with high affinity and spread readily when plated onto surfaces coated with this ligand (
). GD25 cells complemented with β1B (GD25β1B), on the other hand, exhibited reduced binding and a spreading that was limited to distinct peripheral assemblies where β1-integrin co-localized with tyrosine-phosphorylated proteins (
). The finding of the Tyr(P) complexes indicated that the β1B-integrin retained some signaling capacity, and in this study, we have used this to explore the signaling associated with this event of spreading.
EXPERIMENTAL PROCEDURES
Materials—The following antibodies were purchased: mouse anti-FAK2A7 (Upstate Biotechnology, Inc., Lake Placid, NY), mouse anti-paxillin, mouse anti-Crk, rabbit anti-Tyr(P), mouse anti-Tyr(P) (PY20), mouse anti-Rac1, mouse anti-Cdc42, mouse anti-p130Cas, Tyr(P)-RC20:HRPO (Transduction Laboratories), goat anti-DOCK180 (N-19), goat anti-DOCK180 (C-19) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), mouse anti-human VASP (IE273; ALEXIS Biochemicals), mouse anti-talin, mouse anti-human vinculin (Sigma), fluorescein isothiocyanate-conjugated donkey anti-mouse, fluorescein isothiocyanate-conjugated donkey anti-rabbit, lissamine-conjugated donkey anti-goat, rhodamine-conjugated donkey anti-mouse, rhodamine-conjugated donkey anti-rabbit, coumarine-conjugated donkey anti-rabbit, and horseradish peroxidase-conjugated sheep anti-mouse (Jackson Immunoresearch Laboratories Inc.). Polyclonal anti-p130Cas was kindly provided by Dr. J. T. Parsons (Department of Microbiology, University of Virginia, Charlottesville, VA). Alexa Fluor® 488 phalloidin, Alexa Fluor® 568 phalloidin, and wheat germ agglutinin conjugated to lissamine were from Molecular Probes (Leiden, The Netherlands). Other reagents used were PP1 (Alexis Corp.), PP3 (Calbiochem), platelet-derived growth factor (R&D Systems), and the glycine-arginine-glycine-aspartic acid-serine (GRGDS) peptide (Bachem Feinchemicalien AG). The GST-invΔ1 (
) protein was purified according to the protocol provided by the manufacturer (Amersham Biosciences).
Plasmids—Expression vectors encoding GFPp130Cas and GFPΔ SDp130Cas were constructed by insertion of PCR fragments from pRc/CMV-Cas(ctMyc) encoding murine p130Cas (
). To generate GFPΔSDp130Cas, amino acids 117–420 were deleted. GFPCrkII, GFPCrkII SH2 (amino acids 1–110), and GFPCrkII SH3 (amino acids 110–235) were obtained by PCR from pE/L-GFPCrkII encoding murine CrkII and inserted into pCB6GFP-N. The constructs were validated by sequencing. The Rc/CMV-Cas(ctMyc) construct was kindly provided by Dr. S. Hanks (Vanderbilt-Ingram Cancer Center). The pCB6GFP expression vectors, pE/L-GFP-CrkII (
). The cells were grown in Dulbecco's modified eagle medium (DMEM) containing 10% heat inactive fetal bovine serum, l-glutamine (2 mm), penicillin (100 units/ml), streptomycin (100 μg/ml), and puromycin (20 μg/ml). To transfect the cells, they were incubated with a mixture of 3 μl of LipofectAMINE 2000 (Invitrogen) and 0.8 μg of plasmid for 5 h in antibiotic-free conditions, whereafter the medium was replaced and the cells were incubated for 14 h prior to spreading.
Cell Spreading and Immunofluorescence Staining—Cell spreading and immunofluorescence staining were performed as previously described (
). In brief, coverslips were coated with GST-invΔ1 (10 μg/ml) overnight at 4 °C and blocked with 1% heat-treated bovine serum albumin in phosphate-buffered saline at 37 °C for 1 h. Cells were detached and incubated in serum-free DMEM containing cycloheximide (25 μg/ml; Sigma) on bovine serum albumin-blocked Petri dishes for 40 min. Thereafter, the GRGDS peptide (0.1 mg/ml) was added, and the cells were incubated for an additional 20 min. Cycloheximide and GRGDS was used to inhibit the GD25 fibroblasts from synthesizing extracellular matrix proteins and to block binding to αvβ3-integrins, respectively. After this, 2 × 105 cells were allowed to attach on invasin-coated coverslips at 37 °C for 3 h. Unattached cells were washed away, and the remaining cells were fixed in 2% paraformaldehyde for 10 min. The fixed cells were permeabilized with 0.5% Triton X-100 and further processed for double or triple immunofluorescence labeling. Samples were mounted onto microscope slides using ProLong Antifade (Molecular Probes). The pictures were captured using a microscope (Zeiss Axioskop 50) and a CCD camera (ORCA; Hamamatsu) and processed using Adobe software (Adobe).
The inhibition of Src family kinases by PP1 or the negative control, PP3, was performed as described above with the exception that the 60-min pretreatment with cycloheximide was exchanged with a 45-min pretreatment with PP1 or PP3 at various concentrations prior to seeding.
Immunoprecipitation and Western Blotting—Cells were trypsinized, washed once with phosphate-buffered saline, and resuspended in serum-free DMEM containing cycloheximide (25 μg/ml; Sigma) before being plated on GST-invasin (10 μg/ml) or bovine serum albumin. The cells were allowed to attach at 37 °C for 60 min and thereafter were lysed in radioimmune precipitation assay buffer (1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mm NaCl, 50 mm Tris-HCl, 1 mm EGTA, 1 mm Na3VO4, and Complete™ protease inhibitor mixture mix (Roche Molecular Biochemicals)) for 15 min on ice. The lysates were clarified by centrifugation at 14,000 × g for 15 min and precleared with Protein G-Sepharose beads (Amersham Biosciences) coated with mouse γ-globulin (Jackson Immunoresearch Laboratories) for 1 h at 4 °C. After centrifugation, the supernatant was incubated for 3 h at 4 °C with protein G-Sepharose beads precoupled to anti-Crk antibodies. The beads were washed twice with radioimmune precipitation assay buffer and subjected to SDS-PAGE followed by semidry transfer to Immobilon-P transfer membrane (Millipore Corp.). Tyrosine-phosphorylated proteins were detected with anti-Tyr(P) antibodies conjugated with horseradish peroxidase (RC20) and then enhanced chemiluminescence (Amersham Biosciences). The membrane was stripped by incubation in 1 m glycine, pH 2.5, for 30 min, whereafter it was divided and subjected to Western blot with anti-p130Cas or anti-Crk antibodies.
Pull-down—The plasmid-cured Y. pseudotuberculosis strain YPIII (
) was used to stimulate β1-integrins to investigate Cdc42 and Rac1 activation. An overnight culture of YPIII, grown in Luria broth at 26 °C, was centrifuged down, resuspended in antibiotic-free cell culture medium, and incubated for 1 h at 26 °C. This culture was then used to infect a 15-cm dish with cells at 75% confluence (starved for 17 h), at a multiplicity of infection of 5000:1. The infection was carried out at 37 °C in an atmosphere of 5% CO2. The cells were lysed in the pull-down lysis buffer containing 50 mm Tris pH 7.5, 1% Triton X-100, 100 mm NaCl, 15 mm MgCl2, 1 mm EGTA, pH 8.0, and Complete™ protease inhibitor mixture mix. As a positive control for Rac1 activation, the cells were treated with platelet-derived growth factor (3 ng/ml) for 2 min prior to lysis. The lysates were centrifuged at 12,000 × g for 5 min, and the supernatants were saved as the cell lysates. The protein concentration was determined using the Bio-Rad protein assay, and 1300 μg of the cell lysate was incubated with 30 μg of GST-PAK-CRIB (purified as described previously (
)) immobilized on glutathione-Sepharose 4B beads (Amersham Biosciences) for 30 min at 4 °C. The beads were washed four times in wash buffer (10 mm Tris, pH 7.4, 1% Nonidet P-40, 150 mm NaCl, 1 mm EDTA, 12 mm MgCl2), whereafter bound GTP-Rac together with aliquots of cell lysates were subjected to SDS-PAGE (12%) and Western blot with anti-Rac1 antibodies.
RESULTS
β1B-Integrins Have a Partial Signaling and Mediate Formation of Filopodia When Binding to Invasin—To characterize the β1-integrin-mediated spreading, β1A- and β1B-expressing GD25 cells were allowed to spread on invasin-coated plates; triple-stained with phalloidin, wheat germ agglutinin, and anti-Tyr(P) antibodies; and subjected to both fluorescent and phase contrast light microscopy (Fig. 1A). Fluorescent phalloidin and wheat germ agglutinin were employed to visualize the F-actin architecture and cell edges, respectively. Cells expressing β1A-integrins became flat and spread with the typical star-like morphology of fibroblasts where lamellipodia and distinct cell-matrix adhesions were apparent (Fig. 1, A and B). On the other hand, cells expressing β1B-integrins had a round cell body with long extended F-actin structures consisting of fine threads that resembled filopodia, and there were no detectable lamellipodia-like F-actin structures or focal contacts (Fig. 1, A and B). Moreover, the tips of the thin filopodial extensions contained distinct assemblies of tyrosine-phosphorylated proteins (Fig. 1A). There were no such long filopodia-like structures in the GD25β1A cells; the protrusive elements were instead broader lamellipodia-like extensions and some short filopodia-like structures (Fig. 1A). The initial spreading by β1B-integrin-expressing cells was delayed compared with that of cells expressing the β1A variant but was equal after 1 h, where 60% of all attached cells exhibited spread morphology with filopodia or lamellipodia, respectively (Fig. 1B; Supplementary Fig. 1). Maximal spreading of β1B cells was detected after 2 h, but after longer periods of time cells started to detach (Fig. 1B), where very few cells adhered after 6 h (data not shown). Coating density of invasin did not influence the observed spreading behavior; 1–25 μg/ml of invasin resulted in similar spreading for both GD25β1A and GD25β1B cells (data not shown). Upon plating on poly-l-lysine, no spreading at all could be observed (data not shown), which confirmed that the filopodia formed by the β1B-expressing cells was an effect of invasin binding to the β1-integrin receptor. This indicates that the β1B-integrin receptor can mediate spreading with filopodia when binding to invasin but that signaling for lamellipodia formation is impaired. This system therefore provides a unique tool to dissect the complex signaling associated with filopodia formation, which may allow identification of molecular events in spreading that takes place without the formation of lamellipodia. Interestingly, GD25β1B cells that had spread with filopodia and then been ripped off during sample preparation left remnants of the phosphotyrosine-containing tip complexes on the coverslip (Fig. 1C). This shows that the tips of these filopodia are indeed attached to the invasin-coated coverslips, which is supported by our previous finding that the β1-integrins localize to these filopodial tips (
Fig. 1Invasin-stimulated spreading of β1A- and β1B-integrin-expressing cells. GD25β1A cells suspended in and GD25β1B serum-free DMEM, were allowed to spread on GST-invasin-coated glass coverslips for 3 h (A and C) or the indicated times (B). A, cells stained for F-actin (phalloidin), cell membrane (wheat germ agglutinin; WGA), and Tyr(P) (pY). The arrows indicate filopodial extensions, and the arrowheads indicate a protrusive element in GD25β1A. B, attached cells were quantified for spreading and characterized into three groups: cells not spread, cells with only filopodia, and cells having any kind of lamellipodia-like spreading. The values represent percentages of cells in each category, and 65 cells per time were analyzed. C, remnants of Tyr(P)-stained filopodial tip complexes on the coverslip, following ripping off of a GD25β1B cell. D, GD25β1B cells were pretreated with different concentrations of the Src inhibitor PP1 or the negative control PP3 for 45 min prior to spreading on invasin for 3 h. Attached GD25β1B cells were quantified for spreading (filopodia), and the values were normalized to untreated spread cells. The values represent spread cells in relation to control ± S.E. of four separate experiments. Size bars (A and C), 10 μm.
). To investigate whether Src family kinases were important for the observed invasin-promoted filopodia formation by GD25β1B cells, the Src inhibitor PP1 was employed. This inhibitor blocked filopodia spreading of GD25β1B (Fig. 1D) and reduced the amount of detectable Tyr(P) in the tip complexes at lower concentrations (data not shown). As expected, PP1 also blocked spreading of GD25β1A cells (data not shown), whereas the negative control, PP3, did not affect the spreading of the cells (Fig. 1D).
To explore the partial signaling from the β1B-integrin receptor, we aimed to identify proteins present within the Tyr(P) complexes at the filopodial tips. GD25β1B cells spread on invasin were subjected to a screen of immunostaining with antibodies directed against different proteins that are known to localize to focal contacts (Fig. 2A and Supplementary Fig. 2). This screening revealed that VASP and p130Cas were present at sites enriched in Tyr(P), at the tips of filopodia in GD25β1B cells (Fig. 2A). FAK, vinculin, and paxillin were completely absent from these sites and mainly localized to a perinuclear region of the cytoplasm (Fig. 2B), suggesting that the filopodial tip complexes are distinct from focal contacts and that FAK, vinculin, and paxillin are dispensable for filopodia formation. Talin could also be detected in association with the sites enriched in Tyr(P), but the localization was not as distinct as that seen with p130Cas and VASP, since talin also was present in punctate structures along the filopodial extensions (Fig. 2A). However, a closer examination of all detectable filopodial Tyr(P) complexes of the stained cells revealed that the majority of these contained talin (77%) as well as VASP (74%) and p130Cas (82%) (Fig. 2B). In GD25β1A cells that were stained in parallel, all of the focal adhesion markers including FAK, vinculin, and paxillin localized to distal protrusive structures, but also here talin was less distinct (Fig. 2A). Hence, the Tyr(P) complexes at the tips of β1B-mediated filopodia contain only a subset of proteins that can be found in focal contacts.
Fig. 2Filopodia-associated Tyr(P) (pY) complexes in β1B-integrin-expressing cells contain a subset of focal adhesion proteins. GD25β1A or GD25β1B cells suspended in serum-free DMEM were allowed to spread on GST-invasin-coated glass coverslips for 3 h. A, after fixation, the cells were triple-stained for F-actin, Tyr(P), and the indicated proteins. Size bars, 10 μm. The arrows indicate some filopodial tips positive for both Tyr(P) and FC marker protein. B, quantification of the relative amount of distal filopodial Tyr(P) complexes that contain the indicated protein. The values represent the mean percentage ± S.E. of at least 10 cells.
p130Cas-Crk Is Required for Filopodia Formation in GD25β1B Cells—The previous finding that p130Cas was tyrosine-phosphorylated in GD25β1B cells upon adhesion to invasin (
), together with the present observation that p130Cas localized to the tip of invasin-promoted filopodia, suggested a role for this docking protein in filopodia formation. To investigate this, we transfected GD25β1B cells with GFP-tagged full-length p130Cas and a GFP-tagged dominant negative variant of p130Cas (ΔSDp130Cas), which previously has been shown to impair cell migration (
). In nearly 50% of the adherent transfected cells, expression of full-length p130Cas induced formation of lamellipodia-like structures between the bases of extending filopodia, which, as in the corresponding mock-transfected cells, contained distinct peripheral Tyr(P) complexes (Fig. 3, A and F). Interestingly, the expression of ΔSDp130Cas in GD25β1B cells completely blocked the filopodia spreading and assembly of Tyr(P) seen in nontransfected or GFP-transfected GD25β1B cells (Figs. 1 and 3, A and F). This suggests that signaling by p130Cas is crucial for invasin-stimulated filopodia formation by GD25β1B cells. Expression of ΔSDp130Cas also affected GD25β1A cells, which spread less efficiently, where 70% of the cells were more round in shape than corresponding mock-transfected cells (Fig. 3A). However, this effect was less dramatic than the total block seen in GD25β1B cells, where 88% of the transfected cells failed to spread (Fig. 3F).
Fig. 3p130Cas and Crk are required for β1B-integrin-mediated filopodial spreading.A, GD25β1A and GD25β1B cells transfected with GFP-tagged full-length p130Cas, GFP-tagged ΔSDp130Cas, or GFP vector alone (MOCK) were allowed to spread on GST-invasin-coated glass coverslips for 3 h, fixed, and double-stained for F-actin and Tyr(P). B, GD25β1B cells spread on GST-invasin-coated glass coverslips for 3 h and double-stained for Tyr(P) (pY) and Crk or Tyr(P) and Dock180. C, quantification of the relative amount of distal filopodial Tyr(P) complexes that contained Crk or Dock180, respectively. The values represent the mean percentage ± S.E. of at least 10 cells. D, GD25β1A and GD25β1B cells attached to invasin or incubated on bovine serum albumin-coated dishes for 60 min and thereafter lysed in radioimmune precipitation assay buffer. Normalized lysates were immunoprecipitated for Crk, whereafter precipitates and lysates were analyzed by Western blotting using antibodies specific for Tyr(P), followed by stripping of membranes and detection with anti-p130Cas and anti-Crk antibodies. E, GD25β1B cells transfected with GFP-tagged full-length CrkII, GFP-tagged CrkII SH2, or GFP-tagged CrkII SH3 were allowed to spread on GST-invasin-coated glass coverslips for 3 h, fixed, and stained for F-actin and Tyr(P). F, attached and transfected GD25β1B cells were quantified for spreading and characterized within three groups: cells not spread, cells with only filopodia, and cells having any kind of lamellipodia-like spreading. The values represent percentage of cells in each category ± S.E. of three separate experiments. Size bars (A, B, and E) represent 10 μm.
Due to the lack of the substrate domain, ΔSDp130Cas is deficient in binding to the adaptor protein Crk. In analogy with our finding that the p130Cas substrate domain is required for filopodia formation, the p130Cas-Crk scaffold has been shown to participate in cell migration, being important for pseudopodial extension (
). Therefore, immunostainings were performed to explore whether also Crk was associated with the Tyr(P)-rich filopodial tips of GD25β1B cells. The data obtained confirmed our assumption, showing a distinct localization of Crk to the Tyr(P) complexes (Fig. 3, B and C). In addition, Crk also localized to the tips of protrusions in GD25β1A cells (Supplementary Fig. 3B). Moreover, immunoprecipitation of Crk from lysates of nonstimulated and invasin-stimulated GD25β1B cells revealed that adhesion to invasin induced complex formation of Crk with tyrosine-phosphorylated p130Cas, as in GD25β1A cells (Fig. 3D). This was associated with tyrosine phosphorylation of p130Cas (
) and dephosphorylation of Crk, the latter seen as a clear shift in SDS gel migration of Crk from cells stimulated with invasin (Fig. 3D). Dephosphorylation of CrkII has previously been observed to increase the CrkII-p130Cas association and subsequent cell migration (
). To investigate the importance of Crk in β1B-integrin-mediated filopodia formation, GD25β1B cells were transfected with GFP-tagged full-length CrkII, GFP-tagged CrkII Src homology 2 (SH2) domain, or GFP-tagged CrkII SH3 domain 1. Similar to what was seen for p130Cas, expression of the truncated dominant negative variants, the Cas-binding CrkII SH2 domain, or the downstream signaling CrkII SH3 domain 1 caused blockage of filopodial spreading by GD25β1B cells, whereas the full-length variant promoted formation of lamellipodia-like structures in nearly 40% of adherent cells (Fig. 3, E and F).
In addition to binding p130Cas via the SH2 domain, Crk can bind Dock180, which is a guanine nucleotide exchange factor for Rac1, via its first SH3 domain (
). Staining for Dock180 in nontransfected GD25β1B cells revealed that also this signaling molecule was present within the Tyr(P)-rich filopodial tips (Fig. 3, B and C). A weak staining of DOCK180 could also be seen in GD25β1A protrusions, but here DOCK180 was also diffusely spread in the cytoplasm (Supplementary Fig. 3B). Hence, all of the components of this plausible Rac1-activating pathway are present at the filopodial tips of GD25β1B cells.
The Invasin-β1B Interaction Mediates Activation of Rac1— Rac1 has been shown to regulate lamellipodia formation in various cell lines (
). To investigate the involvement of Rac1 during invasin-stimulated spreading, GD25β1B and GD25β1A cells were transfected with GFP-tagged constitutively active (L61Rac1) or dominant negative (N17Rac1) variants of Rac1 before plating onto invasin. The GD25β1A cells were affected as anticipated based on previous work (
) in that expression of L61Rac1 resulted in increased spreading, where all transfected cells formed actin-containing ruffles and were larger than corresponding mock transfectants (Fig. 4A). Expression of N17Rac1 in these cells slightly reduced spreading but had no obvious effect on the cell morphology. A majority (84%) of the L61Rac1-expressing GD25β1B cells formed lamellipodia-like structures similar to that seen upon expression of full-length p130Cas or CrkII (Fig. 3, A, E, and F, and Fig. 4, A and B). There was, however, no inhibitory effect of N17Rac1 on β1B-integrin-mediated filopodia formation (Fig. 4, A and B); rather, there were more filopodia formed, which suggests that this filopodia formation not depends on Rac1.
Fig. 4β1B-integrin-mediated spreading induces activation of Rac1.A, GD25β1A and GD25β1B cells transfected with GFP-tagged constitutively active (L61Rac) or GFP-tagged dominant negative Rac1 (N17Rac) were allowed to spread on GST-invasin-coated glass coverslips for 3 h, fixed, and thereafter double-stained for F-actin and Tyr(P) (pY). Size bar,10 μm. B, attached and transfected GD25β1B cells were quantified for spreading and characterized within three groups: not spread, cells with only filopodia, and cells having any kind of lamellipodia-like spreading. The values represent percentages of cells in each category ± S.E. of three separate experiments. C, GD25β1B cells were infected with plasmid-cured Y. pseudotuberculosis, YPIII, for the indicated periods of times or stimulated with platelet-derived growth factor for 2 min prior to GST-PAK-CRIB pull-down for GTP-Rac1. The pull-downs (upper row) and 20 μg of lysates (lower row) were separated by SDS-PAGE, transferred to polyvinylidene difluoride membrane, and immunoblotted for Rac1. Three individual experiments were performed with similar results.
Given that p130Cas, Crk, and Dock180 co-localized to the Tyr(P) assemblies at the filopodial tips of β1B-expressing cells representing a locally assembled scaffold with a guanine nucleotide exchange factor that can activate Rac1, we decided to evaluate the effect of the invasin-β1B ligation on the activation state of Rac1. To accomplish this, the GST-tagged CRIB of PAK, which interacts with active GTP-bound Rac, was used in a pull-down assay. However, it was not feasible to use adhesion on invasin to stimulate cells for the GST-CRIB-PAK pull-down, since this pull-down assay requires a substantial amount of cell material, which is difficult to obtain when plating cells onto dishes. We therefore took advantage of the specific binding of invasin-expressing bacteria to β1-integrin receptors (
). We have previously shown that the Y. pseudotuberculosis plasmidcured strain YPIII not bind GD25 cells unless they are transfected with β1A- or β1B-integrins (
), showing that this interaction is β1-integrin-specific. Accordingly, YPIII was used to infect GD25β1B cells for different periods of time followed by GST-PAK-CRIB pull-down. Surprisingly, despite the lack of invasin-induced lamellipodia in the β1B-expressing cells, Rac1 became active after invasin stimulation (Fig. 4C). Thus, GD25β1B cells can activate Rac1 upon binding to invasin. This activation does not lead, however, to lamellipodia formation, which suggests that Rac1 either is active at a wrong location or that some additional factor, which the β1B-integrin is unable to recruit, is needed for Rac1 to induce lamellipodia.
β1B-Integrin-mediated Filopodia Formation Is Independent of Cdc42—Previous studies have shown that Cdc42 regulates filopodia formation in certain cell types (
), and we intended to investigate whether this was the case also for invasin-stimulated filopodia formation in GD25β1B cells. GD25β1A and GD25β1B cells were transfected with the GFP-tagged dominant negative (N17Cdc42) or constitutively active (L61Cdc42) Cdc42 before plating on invasin. However, the dominant negative variant did not inhibit the filopodia formation, and neither did the constitutively active variant seem to induce more filopodia in GD25β1B cells, which suggests that these filopodia form independent of Cdc42 activity (Fig. 5, A and B). The N17Cdc42 construct used has been shown to have a dominant negative effect in other situations (
). To exclude possible functional effects from the GFP tag, we also analyzed the effects of expressing another variant of dominant negative Cdc42, but also a FLAG-tagged N17Cdc42 was without effect on invasin-stimulated filopodia formation in GD25β1B cells (data not shown).
Fig. 5β1B-integrin-mediated filopodia formation does not depend on Cdc42 activation.A, GD25β1A and GD25β1B cells transfected with GFP-tagged constitutively active Cdc42 (L61Cdc42) or GFP-tagged dominant negative Cdc42 (N17Cdc42) were allowed to spread on GST-invasin-coated glass coverslips for 3 h, fixed, and thereafter double-stained for F-actin and Tyr(P). Size bar, 10 μm. B, attached and transfected GD25β1B cells were quantified for spreading and characterized within three groups: cells not spread, cells with only filopodia, and cells having any kind of lamellipodia-like spreading. The values represent percentages of cells in each category ± S.E. of three separate experiments.
In this paper, we show that GD25 cells expressing the β1B-integrin splice variant form filopodia-like structures that contain Tyr(P) complexes at the tips without signs of lamellipodia formation. This system was here used to study the molecular events of filopodia spreading in isolation. The Tyr(P)-containing tip complexes, which adhered to the underlying substrate, were found to contain the p130Cas-Crk-Dock180 scaffold, together with talin and VASP. In agreement with the presence of p130Cas, Crk, and Dock180, we found that invasin-stimulated filopodia formation in GD25β1B cells was associated with activation of Rac1 and that p130Cas and Crk are crucial for formation of the filopodia.
Invasin engagement of β1B-integrins induces phosphorylation of p130Cas (
), dephosphorylation of Crk, and complex formation between these two signaling proteins. The p130Cas protein has been implicated in actin cytoskeletal organization through anchoring and/or assembly of actin filaments. Fibroblasts deficient in p130Cas exhibit decreased actin filament formation with disorganized F-actin architecture, and these cells are also affected in migration (
). In analogy, overexpression of p130Cas or Crk is sufficient to induce cell migration, which depends on the coupling of tyrosine-phosphorylated p130Cas with dephosphorylated Crk (
). The p130Cas protein localizes both to the cytoplasm or perinuclear region and to adhesion sites of adherent cells. Intriguingly, stimulation of β1-integrins by invasin effectively recruits p130Cas to newly formed peripheral adhesion complexes, but p130Cas rapidly disappears from this site upon retractile events and is absent from retraction fibers (
). Hence, this observation together with the present finding of p130Cas-CrkII as being crucial for induction of filopodia formation suggests that this complex is important for initial protrusive F-actin processes and might represent a key constituent of such a precomplex.
The p130Cas-Crk scaffold has previously been implicated in activation of Rac1 through binding of Crk to the Rac1 guanine nucleotide exchange factor Dock180. Consistent with the Rac1 activating potential of this scaffold, there was a clear invasin-induced activation of Rac1 in the β1B-expressing cells. However, Rac activation was not required for filopodia formation, indicating that the p130Cas-Crk complex also triggers actin polymerization by a Rac-independent mechanism. Initially, we thought that this could be by Cdc42, but surprisingly the filopodia formed upon invasin stimulation of β1B-integrins were unaffected by expression of dominant negative Cdc42. Thus, it seems likely that the filopodia formed by GD25β1B cells plated onto invasin are regulated by another mechanism. These filopodia, which are uniformly distributed around the cells that appear totally nonpolarized, might differ from those regulated by Cdc42 that are thought to determine cell spreading direction and polarity (
). Moreover, invasin-promoted internalization of Yersinia, which is a Rac1-dependent process, has also been shown to occur independently of Cdc42 in HeLa and COS-1 cells (
), indicating that Cdc42 is dispensable for the cellular responses induced by invasin. Other GTPases than Cdc42 have been shown to induce filopodia formation in cells, and Cdc42 do not mediate filopodia formation in all types of cells. Recently, Aspenstrom et al. (
) showed that overexpression of constitutive active forms of the GTPases Wrch, RhoD, and Rif induced thin filopodia in porcine aortic endothelial cells, whereas overexpression of active L61Cdc42 instead induced lamellipodia. Moreover, the GTPase TC10, which is a member of the Cdc42 family, has also been shown to induce filopodia in cells (
). Hence, speculatively then, TC10 could be responsible for formation of filopodia downstream of Crk in GD25β1B cells. However, the downstream pathway from Crk to filopodia formation is still elusive.
The invasin-induced activation of Rac1 was not associated with formation of any detectable lamellipodia structures. In this situation, Rac1 become GTP-bound due to stimulation of the β1B-integrin, but this is obviously not enough to mediate lamellipodia spreading. On the other hand, transfection with constitutively active Rac1 resulted in lamellipodia-like spreading in GD25β1B cells showing that Rac1 can induce lamellipodia in these cells but fail to do so when it is activated through invasin-β1B-integrin stimulus. The small lamellipodia-like structures formed by the Rac1-transfected GD25β1B cells are probably caused by global activation of Rac1 that occasionally interacts with the right partners, possibly represented by proteins found at the cell periphery, and this spreading is probably occurring independent of signals from the integrin receptor. The inability of GD25β1B cells to form lamellipodia through activation of the integrin receptor suggests that there are factors additional to assembly of the Cas-CrkII-Dock180 scaffold and subsequent Rac1 activation that are required for formation of lamellipodia. This can be a matter of subcellular localization of key components, where some important Rac1 effectors are absent from the β1B-integrin-induced filopodia, whereas the β1A-integrins, which mediate formation of lamellipodia, can recruit these factors and establish the cytoskeletal connections required. However, the possibility cannot be excluded that ligated β1B-integrins, which do not form clusters as the β1A variant (
), generate a signal that is too “weak” for induction of lamellipodia despite activation of Rac 1.
It is noteworthy that the adhesion structure component vinculin, which was missing from the invasin-induced adhesion complexes in β1B-integrin-induced filopodia, has been implicated in lamellipodia formation, since it can mediate activation of Rac1 and also recruit the Arp2/3 complex to newly forming integrin contacts (
). It is therefore possible that the inability of β1B-integrins to recruit vinculin contributes to the defect-spreading behavior, due to reduced localization of the actin assembly apparatus to the peripheral adhesion sites. However, although the failure of β1B-integrins to recruit vinculin is a plausible “missing link” that is responsible for the incomplete spreading, it does not exclude the possibility that additional players, which also are missing from the invasin-β1B-integrin assembled scaffold, are required for lamellipodia spreading.
We show here that the β1B-integrin variant has a partial signaling capacity, but how the signal is transduced from the receptor to p130Cas is, however, still elusive. In β1B-expressing cells, there is no phosphorylation of FAK in response to invasin ligation (
); nor is FAK recruited to filopodia. It is therefore not likely that this kinase is involved in phosphorylation and recruitment of p130Cas, and instead Src family kinases might play a role. Members of this kinase family are activated and relocalized to cell-matrix adhesions early upon integrin stimulation and are involved in formation of cell-matrix adhesions and actin cytoskeleton modulations (
). In accordance with this, the Src family kinase inhibitor PP1 inhibited filopodia spreading by β1B-expressing cells and normal spreading by cells expressing the β1A-integrin.
) elegantly showed that filopodia arises from reorganization of the actin dendritic network, where a distinct signaling complex at the filopodial tips was suggested to drive filopodia formation, wherein fascin promotes filament bundling, and VASP, which is known to localize to filopodial tips (
), mediates filament elongation. VASP decreases the branching of F-actin and increases the rate of actin polymerization, leading to the formation of long nonbranched actin filaments (
) as those seen in filopodia. The initial signals that start this reorganization of the actin network are, however, still elusive. In our study, we (as did Svitkina et al. (
), was also present, and we also show, for the first time, the presence of p130Cas, Crk, and DOCK180 at this location. This tip complex is probably involved in driving filopodia formation and is clearly different from the common adhesive focal complex structures, which, in addition to the above, contain FAK, vinculin, and paxillin. In agreement with the study by Svitkina et al. (
and we show here that the p130Cas-CrkII complex is required. Whether VASP is downstream of the activity of the Cas-Crk complex or in a separate parallel pathway remains to be elucidated. Nevertheless, our data imply that the tip complex indeed serves to drive filopodia formation. In addition, we show that this signaling complex can adhere with β1-integrins to the substrate, which we base on the requirement for invasin and expression of β1-integrins to observe these complexes, the localization of β1-integrins to the complexes (
), and the fact that these complexes remain on the substrate after cells have been ripped off. Talin could be implicated in the adhesive role of the complex, since it is known to be crucial for integrin function by linking it to the cytoskeleton (
), where one of these binding sites is in the membrane-proximal region of the integrin. This region is common for all β1-integrin splice variants and thus also present in β1B. The presence of talin at the β1B-integrin-induced filopodial tips might therefore reflect this binding. Thus, the tip complex of filopodia might serve a dual function, where VASP and the p130Cas-Crk complex are involved in driving filopodia formation. VASP might be involved by its direct activity on the actin polymerization rate, whereas the exact role of p130Cas-Crk might be to mediate activation of a particular GTPase. Talin possibly is involved in activating and anchoring β1-integrins to F-actin even if the role for talin still has to be formally proven.
Our observation that β1-integrin-mediated signaling can be dissected by comparing responses after stimulation of cells expressing β1A- and β1B-integrin isoforms was here shown to be useful to study filopodia formation in isolation. The partial outcome of β1B-integrin ligation is probably due to failure to activate signaling pathways and make cytoskeletal connections. The data presented in this study provide new information about molecular events involved in filopodia formation, but the data also open new questions on mechanisms behind the observed effect. The β1-integrin A and B splice variants have identical membrane-proximal regions in the cytoplasmic tail but differ in the carboxyl-terminal membrane-distal part (
). It therefore remains to be determined whether it is the common region or the unique 12 amino acids of β1B that mediates the observed signal response that results in recruitment of talin, VASP, and the p130Cas-Crk-Dock180 scaffold and activation of Rac1. Neither can it be excluded that the α-chain of the integrin receptor contributes to the observed effects, since α-chains also mediate certain signaling (
). It is also possible that the state of integrin activation and/or the clustering capacity influences the outcome of receptor ligation. However, although these questions remain to be answered, this paper provides new knowledge indicating that filopodial tips adhere to the substrate and that the p130Cas-Crk complex is a crucial player in filopodia formation, which might well represent an initial phase of cell spreading. It is also clear from our data that the membrane-distal 21 amino acids of the β1A-integrin cytoplasmic tail are important for efficient β1-integrin spreading. Among the proteins that have been shown to interact with this region are ICAP-1, Grb-2, Shc, and talin (
). Consequently, directly or indirectly, one or more of these interactors or some proteins yet to be identified are, together with Rac1, important to mediate signaling and cytoskeletal connections necessary for lamellipodia formation.
Acknowledgments
We thank Dr. Martin Gullberg and Dr. Anna Berghard for helpful comments on the manuscript and Dr. Roland Rosqvist for helpful advice with microscopy work. We thank Dr. M. Way for providing pCB6 GFP expression vectors and constructs, Dr. S. Hanks for providing the Rc/CMV-Cas(ctMyc) vector, and Dr. B. Hallberg for providing GST-PAK-CRIB.
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