Article |
Address correspondence to Michael P. Sheetz, Dept. of Biological Sciences, P.O. Box 2408, Columbia University, Sherman Fairchild Center, Rm. 713, 1212 Amsterdam Ave., New York, NY 10027. Tel.: (212) 854-4857. Fax: (212) 854-6399. E-mail: ms2001{at}columbia.edu
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Abstract |
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Key Words: phosphatase; force; src family kinases; focal contact; mechanotransduction
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Introduction |
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Integrins form the transmembrane link between the cytoskeleton and ECM molecules (Giancotti and Ruoslahti, 1999). They are composed of two subunits, and ß, and each
ß combination has its own binding and signaling properties. Interestingly, some integrins can bind to several ECM ligands (Giancotti and Ruoslahti, 1999). The
v/ß3-integrin, originally described as the vitronectin (VN)* receptor, binds to a variety of plasma and ECM proteins, including vitronectin, fibronectin (FN), fibrinogen, von Willebrand's factor, and bone sialic protein 1 (Krutzsch et al., 1999). Most of these ligands encode an RGD sequence that is presumed to represent the binding site for
v/ß3-integrins.
v/ß3-integrins are major components of focal contacts on FN and VN and localize to the tip of focal contacts where centripetal extension occurs (Felsenfeld et al., 1999).
v/ß3-integrins associate with c-Src (Hruska et al., 1995) and the ß3-cytoplasmic tail of
v/ß3-integrins has been shown to mediate activation of c-Src in osteoclasts (Feng et al., 2001).
The family of transmembrane receptor-like protein tyrosine phosphatases (RPTPs) includes a number of proteins sharing the characteristics of an extracellular domain and intracellular PTP-homology domains. RPTPs have been implicated in the regulation of integrin-mediated events (Petrone and Sap, 2000). The RPTP CD45 associates with ß2-integrins and the absence of CD45 enhances the adhesion to FN (Shenoi et al., 1999). In addition, the RPTP LAR localizes to adhesion sites in regions undergoing disassembly (Serra-Pages et al., 1995). Another member of this family that regulates adhesion events is RPTP. Overexpression of RPTP
increases substrate adhesion and prevents cell rounding induced by insulin or EGF (Moller et al., 1995; Harder et al., 1998). More intriguingly, gene inactivation of RPTP
not only delays spreading on FN but also impairs activation of Src family kinases (SFK; Ponniah et al., 1999; Su et al., 1999). No soluble or cell-surfaceanchored ligands are known to bind the extracellular domain of RPTP
(Petrone and Sap, 2000). It has been suggested that RPTP
forms a symmetrical-inhibited dimer in which a helix-turn-helix wedge element from one monomer is inserted into the catalytic cleft of the other monomer (Jiang et al., 2000). Upon activation, RPTP
associates with SFK such as c-Src, Fyn, and c-Yes (Zheng et al., 1992; Harder et al., 1998; Su et al., 1999). RPTP
dephosphorylates the negative regulatory tyrosine phosphate (Tyr529 in murine c-Src), causing c-Src activation (den Hertog et al., 1993; Zheng et al., 2000).
Here, we show that RPTP associates with
v/ß3-integrins during early spreading. In addition, we show that this association is required for the activation of SFK, the assembly of focal complexes, and the strengthening of integrincytoskeleton bonds on both FN and VN. Moreover, we provide evidence that RPTP
is a critical part of an early force-dependent signal transduction cascade.
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Results |
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RPTP transiently colocalizes with
v-integrins
Next, we wanted to determine whether RPTP colocalizes with
v/ß3-integrins. RPTP
-/- cells were transiently transfected with an RPTP
construct in which the D2 phosphatase domain was replaced by YFP (Buist et al., 2000). To confirm functionality, we performed spreading assays with RPTP
-YFP transfected RPTP
-/- cells and found a spreading ability similar to the RPTP
+/+ cells (Fig. 2 B). Indirect immunofluorescence was used to visualize the
v-integrins and paxillin. Confocal microscopy revealed that RPTP
colocalized with
v-integrins and paxillin at the leading edge on both FN and VN 30 min after plating in spreading cells (Fig. 2 A, ae and ko). After 240 min, all cells were spread and RPTP
was diffusely localized in the membrane (Fig. 2 A, fj and pt), whereas colocalization of
v-integrins with paxillin occurred in the area of focal contacts (Fig. 2 A, j and t). On LA, there was no localization of RPTP
to the leading edge in spreading cells (Fig. 2 C). Thus, we find evidence of a transient association between RPTP
and
v/ß3-integrins during the early phases of focal complex formation on both FN and VN.
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RPTP modulates the formation of focal complexes in association with
v/ß3-integrins via activation of Fyn
To study more directly the interplay between RPTP and
v/ß3-integrins, we observed the distribution of GFP-tagged paxillin on different substrates. Paxillin is a focal adhesionassociated adaptor protein that is a known marker for focal adhesions and focal complexes (Beningo et al., 2001). GFP-paxillin distribution in distinct adhesion sites versus cytoplasmic localization was analyzed in spread cells 30 min after plating. In RPTP
+/+ cells on both substrates, GFP-paxillin distributed to peripheral stripes in the majority of cells (Fig. 4, A [a and g] and B). In contrast, in RPTP
-/- cells, there was more than twofold lower focal complex or contact formation at early times. GFP-paxillin remained mainly cytoplasmic and localized to the leading edge (Fig. 4, A [d and j] and B). To determine the function of the
v/ß3-integrin in this process, we plated GFP-paxillintransfected cells on FN and VN in the presence of GPen. In RPTP
+/+ cells on both substrates, there was significantly less formation of distinct adhesion sites with GPen, as judged by paxillin assembly (Fig. 4, A [b and h] and B). In contrast, there was no further reduction in the number of cells exhibiting formation of adhesion sites in the RPTP
-/- cells with GPen (Fig. 4, A [e and k] and B). As expected, GPen drastically reduced the ability of both RPTP
+/+ and RPTP
-/- cells to spread on VN (Fig. 4, A [h and k] and B).
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Loss of RPTP does not affect integrinligand interactions
The sensitivity of cell spreading on ECM substrates to RPTP expression raises the possibility that RPTP
also modulates integrinligand interactions (Hughes and Pfaff, 1998). To study more directly the role of RPTP
in regulating integrinligand interactions, we placed ligand-coated beads on the upper surface of spreading fibroblasts with a laser trap (<0.5 µm from the leading edge of the cell). This allows the quantification of changes in ligand binding and the dynamics of traction force generation by integrins (Choquet et al., 1997), which are parameters that are essential to cell spreading and migration (Lauffenburger and Horwitz, 1996). Beads were coated with a recombinant fragment of FN (FNIII7-10), purified human VN, or BSA. Beads coated with either FNIII7-10 or VN bound to the surface with a similar frequency and significantly better than BSA-coated control beads (Fig. 5 A). The binding of VN-coated beads was reduced to the level of control beads by pretreatment with GPen (Fig. 5 A). When FNIII7-10coated beads were placed and held on the upper surface of cells in the presence of GPen, there was a significant reduction of bead binding on both RPTP
+/+ and RPTP
-/- cells (Fig. 5 A). Together, these results suggest that integrinligand interactions are not modulated by RPTP
activity. The contrast between the impaired cell spreading and the unaffected bead binding indicates that RPTP
may regulate the strength or dynamics of integrincytoskeleton interactions.
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Reinforcement of integrincytoskeleton linkages depends on RPTP,
v/ß3-integrins, and activation of Fyn
To further analyze the reinforcement process, we centrifuged onto cells (5 min/50 g) large beads (5.9-µm diam) coated with either FNIII7-10 or human VN. In contrast to the small beads used for the laser trap experiments, the large beads can cause the establishment of adhesion sites and, therefore, reinforcement by cellular contractions independent of laser trap restraint (Galbraith et al., 2002). Interestingly, 74% of the FNIII7-10coated beads showed accumulation of GFP-paxillin to the bead periphery after 30 min on RPTP+/+ cells (46% of VN-coated beads; unpublished data). In contrast, only 14% of the FNIII7-10coated beads (7% of VN-coated beads; unpublished data) on RPTP
-/- cells showed redistribution of paxillin to the binding site (Fig. 6, A and B). Next, we wanted to examine the function of SFK and
v/ß3-integrins in the reinforcement process. Interestingly, pretreatment with GPen reduced the fraction of FNIII7-10coated beads accumulating paxillin on RPTP
+/+ cells to 18%, but caused no significant reduction in the number of accumulating beads on the RPTP
-/- cells (11%; Fig. 6, A and B). Because the pretreatment with GPen prevented specific binding of VN-coated beads to the upper surface of RPTP
+/+ and RPTP
-/- cells (Fig. 5 A), there was no accumulation of paxillin around VN-coated beads in any case (unpublished data). Coexpression of CSK with paxillin decreased the number of FNIII7-10coated beads, causing accumulation of paxillin to 16% in RPTP
+/+ cells, whereas there was no further reduction in RPTP
-/- cells (8%; Fig. 6, A and B). Consistent results could be obtained with VN-coated beads (8% for RPTP
+/+ cells and 5% for RPTP
-/- cells; unpublished data). We have shown earlier that RPTP
-dependent activation of Fyn is needed for the formation of focal complexes and contacts, suggesting that it might affect reinforcement. Indeed, coexpression of Fyn with GFP-paxillin led to a significant increase in the number of RPTP
-/- cells accumulating paxillin around beads (68%). In clear contrast, the coexpression of c-Src or c-Yes did not increase the number of accumulating beads in RPTP
-/- cells, but coexpression of c-Src reduced the number of cells responding to the beads in RPTP
+/+ cells to 34% (Fig. 6 C). Reintroduction of wild-type RPTP
into RPTP
-/- cells restored the ability to assemble paxillin at the binding site (Fig. 6 B). To confirm ligand specificity, we used Con Acoated beads on RPTP
+/+ cells, which showed accumulation of GFP-paxillin at the site of adhesion in only 7% of the beads, possibly due to unspecific activation of integrin receptors (Fig. 6 B). To exclude the possibility of a volume effect around the beads, we transfected RPTP
+/+ cells with EGFP alone, which did not increase the signal intensity around the beads in any case (Fig. 6 B, bottom right). Together, these results are consistent with the idea that RPTP
- and
v/ß3-integrindependent reinforcement of integrincytoskeleton linkages is mediated by activation of SFK, in particular, activation of Fyn.
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Discussion |
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RPTP is a well-characterized activator of SFK (Ponniah et al., 1999; Su et al., 1999). Although previous studies have suggested that SFKs are not involved in the assembly of focal contacts (Bockholt and Burridge, 1995; Klinghoffer et al., 1999), those results were obtained after several hours of incubation on a substratum, where we also find normal focal contact formation in RPTP
-/- and SYF cells (unpublished data). Our data obtained with coexpression of CSK or with the SYF cells suggest that SFK could transmit a signal for focal complex assembly as well as disassembly. It has been shown recently that SFKs are needed for the maintenance of focal contacts (Li et al., 2002) as well as for the tyrosine phosphorylation of proteins in early focal contacts (Volberg et al., 2001). c-Src has been shown to regulate focal contact turnover (Fincham and Frame, 1998; Felsenfeld et al., 1999). On the other hand, Fyn has been reported to be involved in the organization of the cytoskeleton (Stein et al., 1994; Thomas et al., 1995), and is thought to be responsible for cell volumedependent changes in the phosphorylation of cytoskeletal and focal contact proteins (Kapus et al., 2000). In addition, Fyn has been implicated in Rho-mediated cellcell adhesion events (Calautti et al., 2002). Our data indicate that SFK, although activated at the same time by the same enzyme, can have opposing effects on the formation of focal contacts. Fyn expression reconstituted almost wild-type levels, whereas expression of c-Src reduced the response to force and the formation of focal complexes. These results are of particular interest because they show that the decrease in force responsiveness with RPTP
deficiency can be partially compensated by high levels of Fyn. These data suggest parallel paths of force sensing in structural components (Sawada and Sheetz, 2002), which might be the target of force-dependent, RPTP
-mediated activation of Fyn. Therefore, the role of SFK remains controversial, especially during the very beginning of contact formation, and a dual role of SFK in assembly, as well as disassembly/turnover, of focal contacts has to be taken into account.
Recently, it has been shown that overexpression of RPTP leads to an increased cellsubstrate adhesion that is associated with increased levels of tyrosine phosphorylation of paxillin, c-Src, and/or focal adhesion kinase (Harder et al., 1998). In addition, it has been demonstrated that expression of RPTP
corresponds with a low tumor grade in breast cancer (Ardini et al., 2000). Our results indicate that RPTP
accelerates the assembly of adaptor and structural proteins at adhesion sites, thereby stabilizing the link between integrins and the cytoskeleton. It is probably not RPTP
itself that is interacting with these proteins; rather, RPTP
induces the appropriate signals. Regulated force generation is essential for cell spreading and migration. For spreading to proceed, adhesion sites must be continually remodeled (Sheetz et al., 1999). Our data raise the possibility that the lack of appropriate signals leads to weak linkages and results in an impaired plasticity in the integrincytoskeleton linkages needed for rapid cell spreading.
How cells respond to matrix rigidity through force sensing and convert physical cues to biochemical signals is still largely unknown. Besides the mechanical stimuli leading to enlargement of cell adhesions sites (Riveline et al., 2001), other stimuli affect cell behavior including stretch (MacKenna et al., 1998; Sawada et al., 2001) and flow-induced shear stress. Both transmembrane and cytoskeletal mechanisms of force sensing and force transduction have been postulated (Meyer et al., 2000; Sawada and Sheetz, 2002). In addition, force-dependent unfolding of cryptic proteinprotein interaction sites in matrix molecules (Zhong et al., 1998) and recruitment of integrins, leading to activation of MAPKs (MacKenna et al., 1998), has been suggested. Specifically, v/ß3-integrins have been implicated in the transmission of mechanical signals (Wilson et al., 1995; Goldschmidt et al., 2001). Nonetheless, the mechanisms leading to the transformation of force into a biochemical signal are only poorly understood. The data presented here suggest that RPTP
localizes with
v/ß3-integrins in a complex and that is a critical component in the force-dependent activation of subsequent signaling steps. Although RPTP
has an extracellular domain, which has been shown to interact in cis with other transmembrane proteins (Zeng et al., 1999), there are no known ligands either soluble or cell-surface bound (Petrone and Sap, 2000). RPTP
may homodimerize on the cell surface (Jiang et al., 2000) and the inactive, dimeric form could be activated by the application of force via
v/ß3-integrins, leading to mechanical separation of the dimer. However, the precise mechanism leading to the activation of RPTP
remains speculative.
In mice missing RPTP, there are severe hippocampal abnormalities and learning defects consistent with a developmental deficit in radial neuronal migration (unpublished observation). The similarity with Fyn deficiency (Grant et al., 1992) and the documented role of
3- and
v-integrins in this process (Anton et al., 1999) underlines a possible role for neuronal RPTP
in mechanotransduction at points of glial/neuronal contact during migration of neurons.
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Materials and methods |
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Spreading assays
40 µg/ml LA, 5 µg/ml VN, and the 120-kD chymotryptic fragment of FN (6.6 µg/ml) were adsorbed onto tissue culture plastic and blocked with DME/0.5% BSA. Control substrates were blocked without the application of ECM. Cells were briefly trypsinzed and trypsin was inactivated with 1 mg/ml soy trypsin inhibitor in DME. Cells were washed twice (DME/0.5% BSA) and incubated for 30 min with or without GPen (0.5 mM) or anti-v or anti-ß3 antibodies (25 µg/ml) before plating. Cells were fixed with 2% paraformaldehyde (PFA) and stained with 0.5% toluidine blue in PFA. 20 fields were counted in each well (20x; Olympus). For paxillin distribution assays, cells were transiently transfected using Fugene 6 (3:1) with GFP-paxillin (0.5 µg/ml), CSK (0.5 µg/ml), wild-type Src, Fyn, or Yes (each 0.5 µg/ml) and plated in the presence or absence of GPen. The number of spread cells exhibiting accumulation of GFP-paxillin in distinct adhesion sites (focal complexes or contacts) versus cells having cytoplasmic localization of GFP-paxillin was counted in two independent sets of experiments by independent observers. Pictures were taken using confocal microscopy (100x; Fluoview 300; Olympus).
Immunoprecipitations and Western blotting
For immunoprecipitations, extracellular proteins of 16 x 106 cells were cross-linked in the presence of 2 mM dithiobis succinimidyl propionate for 15 min and lysed on ice in 50 mM Tris-HCl, 150 mM NaCl, 50 mM NaF, 1 mM Na3VO4, 1% NP-40, 1 mM PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin, pH 7.6 (lysis buffer). The lysates were incubated with polyclonal anti- v or anti-
5 antibodies for 2 h at 4°C on a rotating wheel with protein ASepharose beads added for the second hour. Beads were washed twice in lysis buffer and twice in wash buffer (50 mM Tris-HCl, pH 8.0) and resuspended in 2x SDS-PAGE sample buffer. For whole cell lysates, cells were lysed and lysates were diluted in 3x SDS-PAGE sample buffer. Equal amounts of proteins were further analyzed by SDS-PAGE followed by Western blotting using polyclonal anti-RPTP
, anti-
v, anti-ß3, phosphospecific anti-SFK, or monoclonal antic-Src and dephosphospecific anti-SRC antibodies (clone 28) with immunoreactive bands being visualized by ECL detection.
Immunofluorescence staining
Fibroblasts were transfected with EYFP-RPTP or GFP-paxillin (0.5 µg/ml) and plated as described in Spreading assays. Cells were fixed 30 min after plating in 4% PFA for 20 min and permeabilized with 0.2% Triton X-100. Cells were incubated with monoclonal antipaxillin (mouse) and anti-
v antibodies (hamster) and a polyclonal phosphospecific anti-SFK antibody for 1 h followed by detection with Alexa-labeled (488, 568, and 647 nm) secondary antibodies. Samples were further analyzed by confocal microscopy (Fluoview 300; Olympus).
Bead assays
0.91- and 5.9-µm beads were coated as described previously (Felsenfeld et al., 1996). In brief, carbodiimide-derivitized latex beads were coupled with ovalbumin (800 µg per 0.5-ml beads). Beads were derivitized with biotin and coated with avidin neutralite (150 µg per 50-µl beads). Finally, beads were coupled with biotinylated FN (FNIII 7-10; 0.61 µg per µg/µl beads) or VN (0.83 µg per µg/µl beads) and blocked with BSA-biotin (67 µg per 50-µl beads).
For bead binding assays, cells were briefly lifted and plated on LA-coated coverslips. Beads were held for 3 s 0.20.5 µm from the leading edge, using a 100-mW (40 pN/µm) optical gradient laser trap setup (Axiovert 100TV; Carl Zeiss Microlmaging, Inc.) that was calibrated as described elsewhere (Choquet et al., 1997). For MSD assays, ligand-coated beads were held in the laser trap on the cell surface for 30 s or until the bead had moved >500 nm from the trap center. The x and y coordinates were determined from video micrographs using single particle tracking routines performed using Isee Software (Invision Corporation/Silicon Graphics O2). Tracking accuracy was 510 nm for 1-µm beads. The MSD was calculated using an algorithm modified from Qian et al. (1991).
For reinforcement assays using large beads (5.9 µm), cells were transiently transfected with GFP-paxillin, and ligand-coated beads were spun (5 min/50 g) onto the cells. Binding was assessed by confocal microscopy and image analysis using ImageJ (1.24 d; http://rsb.info.nih.gov/ij/). Beads were scored if the signal intensity was more than twice surrounding area within 10 µm from the leading edge. For real-time paxillin recruitment assays, cells were transiently transfected and treated as described above. Beads were held on the cell surface until the bead had moved out of the trap. Pictures were taken using a cooled CCD camera (Coolsnapfx) mounted to the trap set up (model IX70; Olympus) and analyzed using ImageJ.
Materials
Monoclonal anti-v and -ß3 antibodies were from BD Biosciences. Polyclonal anti-
v, anti-
5, and anti-ß3 antibodies were from CHEMICON International, Inc. The monoclonal antipaxillin antibody was from Transduction Laboratories. The anti-Src antibody (Ab327) was from Oncogene Research Products. The phosphospecific (Y416) anti-SFK antibody was from Cell Signaling. The de-phosphospecific (Y527) anti-Src antibody (clone 28) was a gift of H. Kawakatsu (University of California, San Francisco, San Francisco, CA). The polyclonal anti-RPTP
was produced as described elsewhere (Su et al., 1999). The anti-Fyn antibody was from Upstate Biotechnology, the anti-Yes antibody was from Transduction Laboratories. ECL reagent, peroxidase-coupled antirabbit and antimouse IgG antibodies, and protein ASepharose beads were from Amersham Biosciences. Dithiobis succinimidyl propionate was from Pierce Chemical Co. PVDF membranes for Western Blotting were obtained from Millipore. GPen was from Sigma-Aldrich. Alexa red (568 and 647) and Alexa green (488)labeled antihamster, antimouse, and antirabbit IgG antibodies were from Molecular Probes. All beads were from Polyscience. Fugene 6 was from Roche Diagnostics. The plasmid containing the pRK5GFP-paxillin was a gift of K.M. Yamada (National Institute of Dental and Craniofacial Research, Bethesda, MD). pSGRPTP
-516-YFP was provided by J. den Hertog (Netherlands Institute for Developmental Biology, Utrecht, Netherlands). pECMV-CSK was a gift of E.E. Marcantonio (Columbia University, New York, NY). pUSEsrcwt was from Upstate Biotechnology, pCMV5fyn was a gift of M. Resh (Memorial Sloan-Kettering Cancer Center, New York, NY), pMIKwt-yes was a gift of M. Sudol (Mount Sinai Medical Center, New York, NY). All other reagents were of the purest grade available.
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Acknowledgments |
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This work was supported by the National Institutes of Health (to M.P. Sheetz and J. Sap) and the Deutsche Forschungsgemeinschaft (G. von Wichert).
Submitted: 15 November 2002
Revised: 25 February 2003
Accepted: 26 February 2003
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