Conformation, Localization, and Integrin Binding of Talin Depend on Its Interaction with Phosphoinositides*

Véronique MartelDagger §, Claire Racaud-Sultan, Sandra DupeDagger , Christiane MarieDagger , Frédérique Paulhe, Antoine Galmiche||, Marc R. BlockDagger , and Corinne Albiges-RizoDagger

From the Dagger  LEDAC, UMR CNRS/UJF 5538, Institut Albert Bonniot, Faculté de Médecine de Grenoble, 38706 La Tronche Cedex,  Institut Fédératif de Recherche Claude de Preval, INSERM U326, Hôpital Purpan, 31059 Toulouse Cedex, and || INSERM U452, Biologie Cellulaire et Moléculaire des Microorganismes Pathogènes et de leurs Toxines, Faculté de Médecine de Nice, 06107 Nice Cedex 02, France

Received for publication, March 16, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Talin is a structural component of focal adhesion sites and is thought to be engaged in multiple protein interactions at the cytoplasmic face of cell/matrix contacts. Talin is a major link between integrin and the actin cytoskeleton and was shown to play an important role in focal adhesion assembly. Consistent with the view that talin must be activated at these sites, we found that phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-bisphosphate (PI4,5P2) bound to talin in cells in suspension or at early stages of adhesion, respectively. When phosphoinositides were associated with phospholipid bilayer, talin/phosphoinositide association was restricted to PI4,5P2. This association led to a conformational change of the protein. Moreover, the interaction between integrin and talin was greatly enhanced by PI4,5P2-induced talin activation. Finally, sequestration of PI4,5P2 by a specific pleckstrin homology domain confirms that PI4,5P2 is necessary for proper membrane localization of talin and that this localization is essential for the maintenance of focal adhesions. Our results support a model in which PI4,5P2 exposes the integrin-binding site on talin. We propose that PI4,5P2-dependent signaling modulates assembly of focal adhesions by regulating integrin-talin complexes. These results demonstrate that activation of the integrin-binding activity of talin requires not only integrin engagement to the extracellular matrix but also the binding of PI4,5P2 to talin, suggesting a possible role of lipid metabolism in organizing the sequential assembly of focal adhesion components.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Integrin-mediated cell attachment to extracellular matrix triggers signal transduction cascades that regulate numerous complex biological processes including cell proliferation, differentiation, and migration, as well as tissue organization (1). The synthesis of phosphatidylinositol 4,5-bisphosphate (PI4,5P2)1 belongs to these adhesion-dependent signaling events (2, 3). Recently, a physical association was found between the alpha 3beta 1 integrin and PI 4-kinase, an intracellular enzyme that controls one step of PI4,5P2 biosynthesis (4). The PIP 5-kinase was shown to be a partner of Rho A, a member of the Rho family monomeric GTPases that is involved in focal adhesion assembly (5, 6). The activation of both enzymes leads to the production of PI4,5P2. It is a phosphoinositide found in all eucaryotic cells that regulates many important cellular processes, including vesicular trafficking, platelet activation, and organization of the cytoskeleton (7). Furthermore, PI 3-kinase activity was shown to play a major role in cytoskeletal rearrangements occurring during cell motility or platelet spreading and aggregation (8-11). Indeed, p85, the regulatory subunit of PI 3-kinase IA can bind to the focal adhesion kinase, a major protein of focal adhesions (12-14). Polyphosphoinositides control the activities of various actin-binding proteins such as profilin and gelsolin to promote actin polymerization (15, 16) and affect protein structure. For example, PI4,5P2 increases the alpha -helix content of profilin (17) and induces conformational changes of many other proteins (18). Phosphoinositides may also increase or stabilize the formation of higher form oligomers, as suggested by the dimerization of the protein kinase Akt (19). Although PI4,5P2 is hydrophobic, it is not found exclusively in membranes since it can form micelles and bind to cytoskeletal proteins such as alpha -actinin and vinculin (20). Some groups have shown that PI4,5P2 dissociates the head to tail interaction of vinculin (21-23). As a result, the talin-binding site becomes accessible, whereas the actin-binding sites are blocked by the bound PI4,5P2 (24).

A large number of proteins found in focal adhesions bind actin. This suggests that these proteins may act at different stages of focal adhesion assembly. For example talin and vinculin may be involved in focal adhesion formation, whereas alpha -actinin may be more important in maintaining or stabilizing microfilaments attachment in mature focal adhesions (25-27). Talin, a 235-kDa protein of focal adhesions, can be cleaved into a 47-kDa N-terminal globular head domain and a 190-kDa rod domain by the endogenous protease calpain II (28). Investigations over the past few years have suggested that talin is a key protein for cytoskeleton/membrane interaction (for review see Ref. 29). Talin probably functions by interacting with particular structural and regulatory proteins of focal adhesions. The N-terminal domain has been shown to bind to layilin (30), focal adhesion kinase (31), polyphosphoinositides (32), and recently to beta 1 and beta 3 cytoplasmic domains (33). Likewise, the C-terminal domain binds also to the integrin cytoplasmic domain (34, 35) and, in addition, to vinculin (36) and to actin filaments (37, 38). Structural studies have revealed that most of these functions are based on talin operating as a homodimer forming a dumbbell-shaped elongated molecule of 51 nm in length with three characteristic areas of condensed mass (39). Talin binding to integrin as a homodimer provides a potential mechanism by which talin may cross-link integrins (40). The 47-kDa domain contains a segment of 200-220 amino acids homologous to membrane binding regions of band 4.1 and ezrin (41). These proteins are members of the FERM family, which includes a number of proteins that mediate linkage between the cytoskeleton and the plasma membrane (42). Among them are the ERM proteins that bind to the cell surface glycoprotein CD44 and actin filaments. These interactions are greatly enhanced by PI4,5P2 (18). Talin and FERM domain containing proteins share homologous structural domains, and they are therefore proposed to be regulated by similar mechanisms. Since cell adhesion induces a signaling cascade that gives rise to elevated level of phosphoinositides and based on the sequence homologies between talin and other members of the FERM protein family, we have investigated the binding of phosphoinositides to talin and the consequences of these interactions on the protein structure and function.

As described previously, we show that talin/integrin interaction in vitro is dependent on the occupancy of the receptor by extracellular ligand (40), a peculiar property of the integrin also observed in vivo (43). Furthermore, our results demonstrate that the association of talin with PI4,5P2 is a prerequisite for its interaction with engaged beta 1 integrin. Finally our data strongly suggest a possible role of phosphoinositides in the sequential assembly of focal adhesions.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Human talin was purified from outdated platelets from the blood center of Grenoble, France, and purified as described previously (44). For some experiments, talin was biotinylated according the manufacturer's instructions (Amersham Pharmacia Biotech). Fibronectin was extracted from human plasma according to the method of Engvall and Ruoslahti (45). PC, PI, PIP, PI4,5P2, and thrombin were from Sigma. [gamma -32P]Phosphate was from Amersham Pharmacia Biotech. Fibrinogen was from Stago.

Monoclonal antibodies against talin (8d4) and vinculin (VIN 11-5) as well as rhodamine-phalloidin were purchased from Sigma. The K20 monoclonal antibody specific for human beta 1 integrin was from Immunotech. Rabbit anti-talin and anti-beta 1 antisera were raised in our laboratory as described previously (40). For the polyclonal serum L1, a fragment of talin corresponding to amino acids 88-198 was expressed as a N-terminal glutathione S-transferase-tagged fusion protein in bacteria. After purification, this protein was injected into rabbits. The hybridoma of monoclonal antibody TS2/16, directed against human beta 1, was purchased from the American Type Culture Collection (Manassas, VA), and IgG were purified on protein A-Sepharose beads (Amersham Pharmacia Biotech). Goat anti-mouse IgG and goat anti-rabbit IgG coupled to horseradish peroxidase were from Bio-Rad and Jackson ImmunoResearch, respectively.

GFP- and His6-tagged Fusion Proteins-- PH domains were constructed as C-terminal fusions with GFP. The cDNAs encoding human PLCdelta and ARNO were kind gifts of Drs. E. Lemichez (Rockefeller University, New York) and M. Franco (Institut de Pharmacologie Cellulaire et Moléculaire du CNRS, Sophia Antipolis, France). The PH domains of PLCdelta and ARNO (amino acids 1-175 and 262-400, respectively) were polymerase chain reaction-amplified with the following primers to which were added the restriction enzyme sites XhoI and BamHI: ARNO 5'-AAAAAACTCGAGCACGGGAGGGCTGGCTCCTGAAG-3'; ARNO 3'-AAAAAAGGATCCTCAGGGCTGCTCCTGCTTCTTC-5'; PLCdelta 5'-AAAAAACTCGAGCAATGGACTCGGGCCGGGACTTCCTG-3'; and PLCdelta 3'-AAAAAAGGATCCTCACTGGATGTTGAGCTCCTTCAGGAAG-5'. The resulting polymerase chain reaction products were digested and inserted into the vector pEGFP-C1 using the same enzymes (CLONTECH).

The polypeptide corresponding to the beta 1 integrin cytoplasmic domain was produced in the BLR(DE3) pLysS Escherichia coli strain containing the vector pET19b-Cytobeta 1. This construct allows the production of the fragment 752-798 of the beta 1 integrin cytoplasmic domain. This peptide was recognized by a polyclonal antibody raised against a synthetic beta 1 cytoplasmic peptide coupled to keyhole limpet hemocyanin.

Cell Culture-- HeLa and NIH3T3 cells were grown on plastic culture plates at 37 °C in a 5% CO2 atmosphere in minimum essential medium with alpha  modification (alpha -MEM) supplemented with 7,5% fetal calf serum. Cells were harvested with phosphate-buffered saline (PBS) supplemented with 1 mM EDTA and 0.05% (w/v) trypsin.

Transient Gene Expression and Fluorescence Microscopy-- The constructions containing the PH domain of PLCdelta and PH domain of ARNO fused to GFP were transfected using LipofectAMINE reagent (Life Technologies, Inc.) in NIH3T3 cells in culture in adhesion on fibronectin-coated coverslips. About 24 h after transfection, the cells were fixed with 3% paraformaldehyde and then permeabilized with 0.2% Triton X-100 in PBS for 10 min. Non-specific protein-binding sites were blocked by 1 h of incubation with 10% goat serum in PBS at room temperature. Subsequent treatment of the cells was performed as described previously (40). Dilutions of the primary antibodies used were as follows: anti-talin 8d4 (1:100) and anti-vinculin (1:250). Rhodamine-phalloidin was used at 30 ng/ml. Alexa 546-conjugated goat anti-mouse from Molecular Probes (Eugene, OR) was used as secondary antibodies. The cells were mounted in mowiol solution and viewed using a confocal laser scanning microscope (Zeiss LSM 410) or a conventional epifluorescence microscope (Olympus Provis AX 70). A minimum of 100 cells per condition was evaluated for the presence of focal adhesions.

Immunoblotting-- Samples were reduced in Laemmli sample buffer. After boiling for 3 min, proteins were resolved on polyacrylamide SDS gel (SDS-PAGE) and then transferred to nitrocellulose (membrane Hybond-C super, Amersham Pharmacia Biotech) using a semi-dry apparatus (Amersham Pharmacia Biotech). The membrane was blocked overnight at 4 °C in Tris-buffered saline (TBS) containing 5% (w/v) fat-free milk. The proteins were detected by blotting with the appropriate monoclonal or polyclonal antibodies in TBS, 0.1% Tween, 5% fat-free milk, followed by an incubation with either anti-mouse or anti-rabbit IgG antibody coupled to horseradish peroxidase. Detection was achieved using a chemiluminescent probe (ECL, Amersham Pharmacia Biotech).

Phosphoinositide Binding Assay-- Phosphoinositides (100 µl) at 1 mg/ml in chloroform/methanol (1:1) were dried under nitrogen in glass tubes. The dried phosphoinositides were resuspended in 100 µl of PBS.

Approximately 10 µg of purified talin in a 100-µl total volume of PBS were incubated with each phosphoinositide for 30 min at room temperature. The phosphoinositides were pelleted by sedimentation at 300,000 × g for 30 min. The supernatant fraction and the lipid pellet were separated. Then, the distribution of talin between the supernatant (unbound) and pellet (phosphoinositide-bound) fractions was determined by analyzing 2:3 of each fraction on a 10% SDS-PAGE followed by Coomassie Blue staining.

As a control, the same experiment was performed with 10 µg of fatty acid-free BSA (Euromedex).

Liposome Binding Assay-- Analysis of protein-lipid interactions by cosedimentation of talin with large multilamellar liposomes were performed as described previously (46) with slight modifications. Briefly, large liposomes were prepared from 80% PC, 20% PI4P or PI4,5P2, or pure PC in a buffer containing 20 mM Hepes, pH 7.4, 0.2 mM EGTA as described (47). Talin in 20 mM Tris acetate, pH 7.6, 20 mM NaCl, 1 mM EDTA, 1 mM beta -mercaptoethanol was centrifuged prior to the experiments for 20 min at 20,000 × g at 4 °C followed by protein determination. The protein (50 µg) was preincubated for 15 min at 22 °C and subsequently incubated in the presence or in the absence of liposomes (0.5 mg of lipids/ml) for 15 min at 22 °C. The total volume of the samples was 0.2 ml. The fractions were centrifuged at 100,000 × g for 30 min, and the presence of talin in the pellet and in the supernatant was tested by a 10% SDS-PAGE followed by Coomassie Blue staining.

Thrombin Proteolysis-- 1.5 µg of each phosphoinositide was preincubated with 10 µg of purified human talin. After 30 min at room temperature 0.1 unit of thrombin was added. Digestion was carried out at room temperature for 60 min and then stopped with Laemmli sample buffer. Each sample was resolved on a 10% SDS-PAGE, and subsequently transferred onto nitrocellulose and incubated with the rabbit polyclonal antibody L1 or the monoclonal 8d4 antibody. In a control, fibrinogen (1 µg/ml) was preincubated with 1.5 µg of each phosphoinositide at room temperature for 20 min. Then, in aliquot fractions, increasing concentrations of thrombin were added, and the incubation was continued for 30 min. Samples were resolved on a 10% SDS-PAGE and Coomassie Blue-stained.

Cell Adhesion Assay and Lipid Extract Analysis on Talin Immunoprecipitates-- Cell culture flasks (75 cm2, Greiner Labortechnik, Poitiers, France) were precoated with 25 µg/ml fibronectin for 2 h at 37 °C. Flasks were then blocked with fatty acid-free BSA (Sigma) at 10 mg/ml in PBS for 1 h at 37 °C.

Adherent HeLa cells were labeled for 3 h at 37 °C with 0.4 mCi/ml [gamma -32P]phosphate in alpha -MEM supplemented with 7.5% (v/v) inactivated fetal calf serum and then harvested with PBS containing trypsin as described above. They were finally resuspended in the medium used for labeling. Cell adhesion assay was performed using 7.106 cells per flask (3.5 ml of cell suspension) that were added to the fibronectin-coated flasks for different times at 37 °C. The starting point corresponds to cells directly resuspended after labeling in 1 ml of lysis buffer. Adherent cells were scraped onto ice in 1 ml of a lysis buffer containing 20 mM Tris-HCl, pH 8, 137 mM NaCl, 10% glycerol, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 µM pepstatin, 10 µg/ml leupeptin, and 1% (w/v) Triton X-100. After 1 h on ice, sonication (20 kHz for 2 × 10 s), and centrifugation (12,000 × g for 15 min at 4 °C), the soluble fraction was collected and subsequently precleared for 30 min at 4 °C with 50 µl of protein A-Sepharose beads. Precleared suspensions were then incubated overnight at 4 °C with non-immune or anti-talin polyclonal serum. Immune complexes were precipitated with 50 µl of protein A-Sepharose. After 1 h at 4 °C, the beads were washed twice in lysis buffer and twice with PBS.

Phosphoinositides were extracted as described previously (48) and separated by TLC following the procedure established by Pignataro and Ascoli (49). Briefly, the lipid extracts were applied on oxalate/EDTA-impregnated silica gel plates, which were developed twice for 120 min with CHCl3, CH3OH, 9.15 M NH4OH (40:40:15). Individual lanes containing commercial standards PIP or PI4,5P2 were stained with iodine vapors. After exposure of the plates for 2 days, the radioactive spots were visualized and quantitated by a PhosphorImager 445 SI (Molecular Dynamics, Inc).

For protein analysis, 1:10 of anti-talin immunoprecipitates were separated on a 10% SDS-PAGE and blotted onto nitrocellulose with the monoclonal antibody 8d4. Quantification of the different bands was performed by a densitometric analysis (NIH Image version 1.62).

Solid-phase Binding Assay of Talin to beta 1 Peptides-- A 47-amino acid peptide corresponding to the cytoplasmic domain of beta 1 integrin was produced as a fusion protein containing a polyhistidine tag (Novagen vector). Microtiter plates (96 wells, MaxiSorp Immuno Plate, Nunc) were coated overnight at 4 °C with 10 µg of peptides per well in a final volume of 200 µl in PBS and subsequently blocked with 1% fatty acid-free BSA in PBS, 1 h at room temperature. Talin purified from human plasma (10 µg per assay) or the 47-kDa domain head of talin, corresponding to amino acids 1-417 (synthesized in vitro using a cotranscription/translation system from Promega) was preincubated with 10 µg of each phosphoinositide in a final volume of 200 µl in PBS. After 30 min at room temperature, talin or the in vitro synthesized 47-kDa domain was added to peptide-coated wells, and the incubation was continued for 1 h at 37 °C. The wells were then washed with PBS containing 1% fatty acid-free BSA, and bound protein was removed by the addition of 30 µl of Laemmli sample buffer (44) followed by incubation of the microtiter plate at 95 °C for 7 min. For each experimental condition, two wells were pooled. Recovered samples were then separated by SDS-PAGE and transferred to nitrocellulose. Talin was then detected by immunoblotting. The presence of the 47-kDa head domain of talin was revealed by autoradiography.

The amount of beta 1 peptides coated in the wells was estimated by ELISA with a polyclonal antibody directed against the cytoplasmic domain of beta 1 integrin.

In Vitro Talin/Integrin Binding Assay-- Microtiter plates were coated overnight at 4 °C with 200 µl of fibronectin at 25 µg/ml or with 100 ng of Ts2/16 or K20 monoclonal antibodies per well. The wells were washed twice with PBS and blocked with 300 µl of 3% fatty acid-free BSA in PBS for 2 h at room temperature. HeLa cells were harvested from culture flasks with trypsin/EDTA and resuspended in alpha -MEM supplemented with 7.5% fetal calf serum (v/v). After two washes with PBS at 4 °C, cells were lysed for 1 h at 4 °C in PBS containing 1% Triton X-100, 1 mM MgCl2, 1 mM CaCl2, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µM pepstatin. The cell lysate was clarified by centrifugation at 10,000 × g for 15 min at 4 °C. The amount of proteins in the lysate was determined by the micro-BCA protein assay (Pierce). Then 500 µg of cell lysate were incubated with coated wells for 90 min at 4 °C. Unbound proteins were removed by three washes with PBS containing 1% fatty acid-free BSA. Purified human talin preincubated 30 min at room temperature with phosphoinositides (50 µg/ml) was added at the concentration of 5 µg per well in PBS, and the incubation was continued for 1 h at room temperature. After 4 washes, bound complexes were removed by the addition of 30 µl of Laemmli sample buffer (44) followed by incubation of the microtiter plate at 95 °C for 7 min. Recovered samples were then separated by SDS-PAGE (7.5% acrylamide) and transferred to nitrocellulose. For each experimental condition, two wells were pooled.

The amount of beta 1 integrin was estimated by ELISA with a polyclonal antibody directed against the cytoplasmic domain of beta 1 integrin.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Interaction of Talin with Phosphoinositides-- Isenberg et al. (50) have previously shown that talin coprecipitates with vesicles composed of PIP or PI4,5P2. However, they performed their experiments with liposomes containing 20% PIP or PI4,5P2. Since conflicting results are often obtained with phosphoinositides/proteins interactions, depending on the use of pure phosphoinositides or membrane associated phosphoinositides, it was therefore important to verify how talin was able to interact with these phosphoinositides under these two packaging conditions.

Association of talin with phospholipid was assessed by a sedimentation assay. Pure vesicles of PC, PI, PIP, or PI4,5P2 were incubated with talin and pelleted by ultracentrifugation. Under these experimental conditions, the lipids were pelleted together with proteins suggesting an association between these two components (51, 52). The presence of talin in recovered supernatants and pellets was analyzed after SDS-PAGE by scanning the corresponding bands visualized after Coomassie Blue staining (Fig. 1A). As a control, the protein solution was also centrifuged without added phosphoinositides. This control allowed us to check that talin sedimentation was not due to aggregation or oligomerization of the protein.


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Fig. 1.   Cosedimentation of talin with phosphoinositides. A, talin (10 µg) was incubated for 30 min at room temperature in the presence of pure vesicles of PC, PI, PIP, PI4,5P2 or as a control without lipids (PBS). After centrifugation at 300,000 × g for 30 min, the presence of talin in pellets (P) and in supernatants (S) was analyzed by Coomassie Blue staining after sample separation on a 10% SDS-PAGE. B, the same experiment was performed with 10 µg of fatty acid-free BSA. Samples were resolved by SDS-PAGE (10%), and the gel was stained by Coomassie Blue. A and B correspond to densitometric quantification of protein bands. The figure illustrates one representative experiment of four performed with similar results. C, talin (50 µg) was incubated for 15 min at 22 °C with large multilamellar liposomes as described under "Experimental Procedures." After centrifugation, the presence of talin in pellets (P) and in supernatants (S) was analyzed by Coomassie Blue staining after sample separation on a 10% SDS-PAGE.

Fig. 1A shows the results of the lipid binding assay. Sedimentation in the ultracentrifuge was examined under five conditions as follows: without any phospholipid, or with PC, PI, PIP, and PI4,5P2, respectively. Although no talin was pelleted in the absence of lipid, and ~30% in the presence of PC or PI, sedimentation reached nearly 98% with PIP and 60% with PI4,5P2. Interaction between talin and phospholipids has been shown previously (53, 54) and is consistent with the result obtained with PC. In the presence of PIP and PI4,5P2, a greater amount of talin was recovered in the pellet.

A control experiment was carried out by replacing talin with BSA. As shown in Fig. 1B, there was no significant cosedimentation of this protein with phosphoinositides, indicating that the talin/phosphoinositide interaction was specific.

As a measure of bilayer association, cosedimentation of talin with large liposomes containing either pure PC or a mixture of PC (80%) and PI, PI4P, or PI4,5P2 (20%), respectively, was analyzed (Fig. 1C). Binding of talin to lipid bilayer was only observed with PI4,5P2 containing liposomes, indicating a restricted specificity of the interaction.

Our results clearly show that under the experimental conditions used, human purified talin bound preferentially to the pure phosphoinositides PIP and PI4,5P2, whereas this interaction was restricted to membrane-associated PI4,5P2.

Association of Talin with Phosphoinositides Occurs in Vivo and Is Modulated during Cell Adhesion-- Earlier studies have shown that overexpression of a cloned type I PIP 5-kinase induced actin polymerization in vivo (55). On the other hand, microinjection of an antibody to PI4,5P2 inhibited the formation of actin stress fibers induced by growth factors (23). Finally, overexpression of a PI4,5P2 phosphatase, an enzyme which converts PI4,5P2 to PIP, disrupted actin stress fibers (56).

To determine the physiological relevance of the interactions of PI, PIP, or PI4,5P2 with talin observed in vitro, phosphoinositides of adherent HeLa cells were prelabeled to equilibrium with 32P. After resuspension by trypsin/EDTA treatment, the cells were plated for different times on fibronectin before immunoprecipitation of talin and extraction of lipids as described under "Experimental Procedures." The amount of talin immunoprecipitated varied for each time of plating due to the increase of the number of adherent cells. Therefore, for each time point of experiment, the amount of immunoprecipitated talin from cell lysates was quantified by Western blotting using an anti-talin monoclonal antibody. The specific phosphoinositides binding to talin was normalized to a constant amount of talin for each experimental time of spreading. Nonspecific binding of phosphoinositides to the beads was estimated on a parallel experiment in which the anti-talin polyclonal antibody was replaced by the non-immune serum. In this case only small amounts of lipids were detected without any significant variation during the time course of the kinetic of adhesion (data not shown). Our results showed that talin coprecipitates with PI and PIP when HeLa cells were resuspended (Fig. 2, A and B), whereas PI4,5P2 became transiently associated with talin after 10 min of adhesion on fibronectin (Fig. 2C). This association of talin with PI4,5P2 observed after 10 min of adhesion parallels phosphoinositide turnover, since it is known that adhesion to fibronectin via integrins results in the accumulation of the PIP 5-kinase product PI4,5P2 (2). On the other hand, the amount of PI and PIP bound to talin quickly decreased to background levels after 25 min of adhesion on fibronectin. Thus, cell adhesion on fibronectin may modulate the association of phosphoinositides with talin and therefore influence its function in focal adhesions.


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Fig. 2.   Association of talin with phosphoinositides varies during cell adhesion on fibronectin. A-C, HeLa cells were labeled with [32P]orthophosphate and kept in suspension (time 0) or plated on a fibronectin matrix for different times (for 5-60 min). After cell lysis, immunoprecipitation with a polyclonal serum against talin or with a non immune serum was performed. Lipids were then extracted from the immunoprecipitate and separated by TLC as described under "Experimental Procedures." For each point, the content of each lipid is expressed after normalization of talin content. For each phosphoinositide, 100% corresponds to the maximum value obtained. A represents the result obtained for PI; B, for PIP; and C, for PI4,5P2. Each graph represents means ± S.D. from three independent experiments.

Phosphoinositide Binding Induced an Apparent Conformational Change in Talin-- Several studies have shown that phosphoinositide binding can modify the structure of proteins (18, 21, 57). In order to determine the effect of phosphoinositide binding on the structure of talin, this protein was submitted to a limited proteolysis with thrombin after a preincubation with each phosphoinositide. It was reported previously that digestion of talin by thrombin leads to the production of two fragments corresponding to the N-terminal globular head domain and to the C-terminal rod domain of talin (58). The digestion pattern was analyzed by immunoblot with the polyclonal serum L1 raised against the 47-kDa head domain of talin (Fig. 3A) or with the monoclonal antibody 8d4 directed against the 190-kDa rod domain of talin (Fig. 3B). As expected, the polyclonal antibody L1 recognized intact talin (Fig. 3A, lane 1) and the globular head domain of talin obtained after thrombin digestion (Fig. 3A, lane 2). Conversely, the monoclonal antibody 8d4, did not recognize at all the head domain of talin but strongly detected the full length and some degradation products of the 190-kDa fragment (Fig. 3B).


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Fig. 3.   PI, PIP, and PI4,5P2 binding induces a conformational change in talin. Talin (10 µg) was subjected to proteolysis by thrombin in the presence (lanes 3-5) or in the absence (lane 2) of phosphoinositides. In the control, no thrombin was added (lane 1). After 60 min at room temperature, samples were analyzed by gel electrophoresis on a 10% SDS-PAGE and immunoblotting. A, the digestion pattern was analyzed with the polyclonal antibody L1 specific for the N-terminal head domain of talin. B, fragments of talin were detected with the monoclonal antibody 8d4 against the 190-kDa tail of talin. The figure illustrates one representative experiment of four performed with similar results. C, as control, fibrinogen (1 µg/ml) was digested by increasing concentrations of thrombin for 30 min at room temperature after preincubation with PI4,5P2 for 20 min at room temperature. After the digestion, the samples were resolved on a 10% SDS-PAGE, which was Coomassie Blue-stained. The last lane contains protein markers.

Fibrinogen, a physiological substrate of thrombin, was digested by different concentrations of the protease after preincubation with PI4,5P2. This control indicated that the presence of PI4,5P2 did not affect the proteolytic activity of thrombin monitored by the shift in the apparent molecular weight of fibrinogen alpha  and beta  chains (Fig. 3C). However, our experiment showed that the presence of phosphoinositides and especially PI4,5P2 greatly enhanced talin susceptibility to proteolysis by thrombin (Fig. 3, A and B, lanes 3-5) as compared with the conditions without lipid (Fig. 3, A and B, lane 2). It is noteworthy that only the rod domain of talin was cleaved, whereas the globular head remained intact, indicating that it keeps a protected structure.

Since PI4,5P2 did not affect the proteolytic activity of thrombin (Fig. 3C), the altered susceptibility of talin to proteolysis was inferred to represent a conformational change in the protein induced by phosphoinositide binding.

PIP and PI4,5P2 Enhance Talin/Integrin Interaction-- Since the association of talin with phosphoinositides leads to a conformational change of this protein, we examined whether this association had any functional consequences. In particular, whether phosphoinositides modulated the association of talin with one of its known partners. Talin is known to interact in vitro with the cytoplasmic domain of integrins beta 1, beta 3, and alpha IIb (34, 35, 59). However, this interaction occurs solely when the integrins are occupied with their extracellular ligands (40, 43). To investigate the role of phosphoinositides in the binding of talin to beta 1 integrins, we examined the ability of the integrin to interact with talin preincubated or not with phosphoinositides in a solid phase assay.

In the first experiment we used peptides corresponding to the cytoplasmic domain of the beta 1 integrin subunit (amino acids 752-798) expressed in bacteria as a fusion protein to a polyhistidine tag. A microtiter plate was coated with these peptides. After three washes and a subsequent blockade with BSA, purified talin preincubated with each phosphoinositide was added. After 60 min at 37 °C, the plate was washed, and the binding of talin was analyzed by immunoblot, after recovering of bound proteins. Under these conditions, talin was shown to bind to the cytoplasmic domain of beta 1 integrin (Fig. 4A, lanes 4 and 5). However, the preincubation of talin with phosphoinositides PIP or PI4,5P2 greatly enhanced its association with the cytoplasmic domain of the beta 1 integrin (Fig. 4A, lanes 6 and 7), whereas it has no effect on the coating of beta 1 in the wells (Fig. 4B) The presence of talin in the well was specifically due to its interaction with the cytoplasmic domain of the beta 1 integrin, since talin was not retained when this peptide was replaced with BSA (Fig. 4A, lane 1). Furthermore, the strong signal obtained with PIP or PI4,5P2 was not due to an aggregation of talin, which leads to its aspecific retention in the wells, since no talin was detected if it was preincubated with PI4,5P2 and subsequently added to a BSA-coated well (Fig. 4A, lane 2).


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Fig. 4.   Binding of talin to immobilized peptides corresponding to the beta 1 cytoplasmic domain. A, microtiter plate was coated with 10 µg of the peptide corresponding to the beta 1 cytoplasmic domain. After blocking with fatty acid-free BSA, human purified talin (10 µg per well) preincubated with phosphoinositides was allowed to bind to the peptide-coated wells for 1 h at 37 °C. After several washes, bound talin was recovered, and the presence of talin was analyzed by immunoblot with the monoclonal antibody 8d4. As a control, 1 µg of purified talin was resolved on the same gel. B, ELISA using a polyclonal antibody directed against the cytoplasmic domain of beta 1 integrin has been performed to check that equal amounts of beta 1 peptides have been coated in each well. The amount of beta 1 peptides does not change regardless of the phosphoinositides added. The figure illustrates one representative experiment of four performed with similar results.

Talin was also reported to interact with both the cytoplasmic domains of alpha  and beta  integrin subunits (35). Consequently, it was important to perform the same experiment with the whole receptor. Lysates of HeLa cells were incubated in fibronectin-coated wells to retain fibronectin receptors. After 3 washes, purified talin preincubated with each phosphoinositide was added, and then the bound proteins were recovered as described under "Experimental Procedures." The presence of talin was analyzed by immunoblot. Fig. 5A shows that in the absence of phosphoinositides or in presence of PI only small amounts of talin were detected (Fig. 5A, lanes 4 and 5). In contrast, the presence of PIP or PI4,5P2 greatly enhanced talin/integrin association (Fig. 5A, lanes 6 and 7). Furthermore, the presence of talin in the wells was specific to fibronectin receptors, since no talin was retained in the wells where fibronectin was replaced with BSA (Fig. 5A, lane 1) or when the preincubation with the cell lysate was omitted (Fig. 5A, lane 2). The presence of lipid did not modify the amount of beta 1 integrin in the wells (Fig. 5A').


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Fig. 5.   Phosphoinositides modulate the interaction between talin and beta 1 integrin. Microtiter plates were coated with 25 µg/ml fibronectin (A) or with 100 ng of K20 (B) or Ts2/16 monoclonal antibodies (C). As a control a coating with 3% of fatty acid-free BSA (A) or with 100 ng of a control IgG2a was performed (B and C). 500 µg of a lysate from HeLa cells was then incubated in coated wells to immobilize integrins. After three washes, 5 µg of purified talin preincubated or not with phosphoinositides was added as indicated. One hour later, after the washes, bound proteins were recovered and separated on a 7.5% SDS-PAGE, and the presence of talin was analyzed by immunoblot with the monoclonal antibody 8d4. As a control for the immunoblot, 0.4 µg of purified talin (A and B) was resolved on the same gel. For each experiment, ELISA using a polyclonal antibody directed against the cytoplasmic domain of beta 1 integrin has been performed to check that equal amounts of beta 1 integrin have been retained in each well (A', B', and C'). The amount of beta 1 peptides does not change regardless of the phosphoinositides added. The figure illustrates one representative experiment of four performed with similar results.

In order to verify that talin was retained in wells via its interaction with integrins, and to analyze whether this interaction in the presence of lipids was dependent on the conformational state of the integrins, the same experiment was performed with specific anti-beta 1 antibody-coated wells. Two different antibodies were used as follows: either the monoclonal antibody K20 that recognizes an extracellular epitope of the beta 1 chain without affecting ligand binding function but having negative signaling effects (60), or the monoclonal antibody Ts2/16, an activating antibody that simulates ligand binding (61). As shown in Fig. 5C, the amount of talin recovered in Ts2/16 mAb-coated wells was greatly enhanced in the presence of PIP or PI4,5P2 (Fig. 5C, lanes 6 and 7), similarly to what was observed with fibronectin-coated wells. With the monoclonal antibody K20 talin was not retained (Fig. 5B, lanes 4-7), indicating that talin/integrin interaction was still dependent on integrin engagement, even in the presence of added PIP or PI4,5P2. Again it is noteworthy that phosphoinositides did not modify the amount of bound beta 1 integrins in the wells (Fig. 5, B' and C').

These latter results were in good agreement with in vitro (40) and in vivo data (43) showing that the interaction between talin and integrins requires integrin occupancy. Furthermore, the same results were obtained with integrins from NIH3T3 cells, a fibroblast cell line (data not shown).

The hypothesis of conformational change of talin upon binding of phosphoinositides was greatly strengthened by testing the binding of the head talin domain to the immobilized cytoplasmic domain of integrin (Fig. 6). The head of talin binds very easily to the cytoplasmic domain of beta 1 integrin, and its binding is specific since the head of talin is not retained when this peptide was replaced with BSA. However, preincubation with phosphoinositides had no further effect on the interaction between talin head and the cytoplasmic domain of beta 1 integrin. This suggests that the effect of phosphoinositides on interaction between whole talin and integrin reflects a conformational change of talin.


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Fig. 6.   Phosphoinositides have no effect on the binding of talin head with the cytoplasmic domain of beta 1 integrin. A, in vitro synthesized 47-kDa head of talin was preincubated or not with phosphoinositides and then was allowed to bind either to BSA-coated wells (lanes 1-4) or to the cytoplasmic domain beta 1-coated wells (lanes 5-8). After several washes, bound proteins were recovered with Laemmli buffer and loaded on a 10% acrylamide gel before analyzing the presence of the 47-kDa head of talin by autoradiography. Lanes 1 and 5, 2 and 6, 3 and 7, and 4 and 8 correspond to the preincubation of talin head without phosphoinositides, with PI, with PIP, and with PI4,5P2, respectively. ELISA using a polyclonal antibody directed against the cytoplasmic domain of beta 1 integrin has been performed to check that equal amounts of beta 1 peptides have been coated in each well. The amount of beta 1 peptides does not change regardless of the phosphoinositides added (not shown). B, the part of autoradiography showing the interaction of the head of talin with the beta 1 peptides (lanes 5-8 of A) was analyzed by scanning densitometry and quantitated using NIH image. The figure illustrates one representative experiment of two performed with similar results.

Binding of PI4,5P2 on Talin Increases Its Affinity for beta 1 Integrin-- Our previous experiments favored the view that PI4,5P2/talin interaction induced a conformational change in the protein that allowed its binding to the cytoplasmic domain of the engaged beta 1 integrin. Whether this conformational change led to an increased in affinity for the integrin or to the oligomerization level of talin that resulted in a larger amount of talin bound to the integrin remained an open question that should be addressed.

In the first experiment, a microtiter plate was coated with increased amounts of biotinylated talin preincubated or not with PI4,5P2. After 1 h of incubation, direct quantification of the adsorbed talin was performed with horseradish peroxidase-labeled streptavidin. Fig. 7A shows that identical concentrations of talin introduced in the wells resulted in the same amount of coated talin in the presence or in the absence of PI4,5P2. This result strongly suggests that PI4,5P2 binding does not promote talin oligomerization. Indeed, such an oligomerization would result in an increase in the amount of coated talin on the plastic.


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Fig. 7.   PI4,5P2 does not affect talin oligomerization but its affinity for beta 1 integrin. A, increasing amounts of biotinylated talin preincubated or not with PI4,5P2 were used to coat plastic wells of a 96-well tray. Subsequently, after the washes and a blocking step, talin was detected with horseradish peroxidase-conjugated streptavidin. B, a constant amount of HeLa cells lysate (400 µg) was allowed to attach to wells coated with Ts2/16 antibodies (100 ng/well). After a blocking step, increasing amounts of talin preincubated or not with PI4,5P2 was added as indicated. After the washes, bound proteins were recovered, separated on a 7.5% SDS-PAGE, and the presence of talin was analyzed by immunoblot with the monoclonal antibody 8d4. Graphs are obtained after analysis of anti-talin immunoblots by scanning densitometry and quantification using NIH Image.

In order to estimate the effect of PI4,5P2 on affinity of talin for beta 1 integrin, we performed the previous solid phase assay using immobilized beta 1 integrin from HeLa cell lysate with Ts2/16 antibodies. This time, increasing amounts of talin preincubated or not with PI4,5P2 were added. After 1 h at room temperature, the plate was washed, and the binding of talin was analyzed by immunoblot after recovering the bound proteins. The quantification of immunoblot showed that the interaction between talin and integrin is dose-dependent (Fig. 7B). Moreover, with PI4,5P2, a saturable binding of talin to beta 1 integrin was observed. Conversely, in the absence of PI4,5P2, this interaction was significantly weaker and did not reach saturation with the talin concentrations used. These curves clearly show a decrease in the apparent Kd value of the talin/beta 1 interaction when PI4,5P2 was present.

PI4,5P2 Regulates Talin Localization in Focal Adhesions-- Until recently, it has been difficult to visualize directly PI4,5P2 near the cell surface. Now, effective reagents for localization of PI4,5P2 within cells have become available with the discovery of pleckstrin homology (PH) domains that bind selectively this phosphoinositide (62). Most PH domains were found to bind phosphoinositides with high affinity but low selectivity, but the PH domain of PLCdelta was shown to bind PI4,5P2 integrated into lipid membranes specifically and with high affinity both in vitro (63, 64) and in vivo (65, 66). On the other hand, the PH domain of ARNO was demonstrated to bind with a high degree of specificity to PI3,4,5 P3 (67, 68). Thus, these PH domains have turned out to be a powerful tool for (i) determining the subcellular localization of these lipids and (ii) specifically sequestering these lipids in order to observe the consequences on protein localization.

We investigated the role of PI4,5P2 in the cellular distribution of talin by transiently expressing either the ARNO or PLCdelta PH domains fused to GFP in NIH3T3 cells. These cells were chosen for their ability to form well characterized focal adhesions. Transient expression of either GFP alone or the ARNO PH domain fused to GFP did not significantly affect talin localization. No accumulation of the GFP-ARNO PH fusion protein at the plasma membrane was observed. Conversely, after the same period of 16 h required for expression, many of the cells transfected with the GFP-PH(PLCdelta ) gained a more rounded appearance and lost completely their substrate attachment for longer expression periods. When the cells were still attached to the substratum at the shorter times of expression of GFP fused to the PH(PLCdelta ), talin was excluded from plasma membrane (Fig. 8A), whereas vinculin was still present in focal adhesions (Fig. 8B) and actin could still be visualized as stress fibers (Fig. 9). A statistical analysis was performed by scoring the talin-positive focal adhesions in a minimum of 100 cells per condition. Fig. 10 shows that more than 80% of cells were negative for talin localization in focal adhesions when the PH domain from PLCdelta protein was expressed, whereas vinculin localization at the tips of actin stress fibers still remained. At this time of expression, the absence of talin in cell contact areas with the extracellular matrix was not due to the disappearance of focal adhesions since vinculin (Fig. 8B), actin (Fig. 9), and integrins (data not shown) showed no difference in their distribution in cells overexpressing the PH domain of PLCdelta or ARNO. So, when the plasma membrane PI4,5P2 is sequestrated by GFP-PLCdelta , talin lost its ability to stay in focal adhesions although these structures were still present. However, under these conditions, the stability of focal adhesions might be impaired. This could account for the rounding up of the cells after longer times of transfection. Finally, our results strongly suggest that PI4,5P2 controls talin localization. This observation may be relevant to the physiological mechanism of focal adhesion disassembly.


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Fig. 8.   PI4,5P2 is necessary for the maintenance of talin in focal adhesions. Vectors encoding the indicated GFP fusion proteins or GFP alone were transiently transfected in NIH3T3 cells in culture on fibronectin-coated coverslips. About 24 h after transfection, the cells were fixed, permeabilized, and processed for immunofluorescence with antibodies against talin (A) or vinculin (B). Detection was done with Alexa 546-conjugated goat anti-mouse IgG secondary antibodies. Arrowheads indicate the focal adhesions. Note that overexpression of GFP, GFP-ARNO/PH domain, or GFP-PLCdelta /PH domain has no effect on the distribution of vinculin (B). On the contrary, overexpression of GFP-PLCdelta /PH domain leads to the disappearance of talin in zones of cell/extracellular matrix contacts (A, arrows). However, this effect is specific for the sequestration of PI4,5P2, since overexpression of GFP-ARNO/PH domain has no effect on talin distribution (A, arrowheads). The figure illustrates one representative experiment of four performed with similar results.


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Fig. 9.   Effect of sequestration of PI4,5P2 on actin stress fibers. Vectors encoding the indicated GFP fusion proteins or GFP alone were transiently transfected in NIH3T3 cells for 24 h. The cells were then fixed and processed for immunofluorescence with rhodamine-phalloidin. Note that actin kept stress fiber structures even if PI4,5P2 is sequestrated by expressing the PH domain of PLCdelta . The figure illustrates one representative experiment of four performed with similar results.


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Fig. 10.   Quantification analysis of focal adhesions in HeLa cells after transfection of PLCdelta - or ARNO-PH domain. Vectors encoding the GFP/PH domain fusion proteins or GFP alone were transiently transfected in NIH3T3 cells in culture on fibronectin-coated coverslips. About 24 h after transfection, the cells were fixed, permeabilized, and processed for immunofluorescence with antibodies against vinculin (VINC), talin (TAL), or actin (ACT) as described under "Experimental Procedures." A statistical analysis has been performed by evaluating for the presence of vinculin- or talin-positive focal adhesions and for the presence of actin stress fibers for a minimum of 100 cells per condition. Quantification has been performed in cells transfected with vectors encoding GFP (white bars), GFP-PH/ARNO (gray bars), or GFP-PLCdelta (black bars). Results are representative of three separate experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Talin is a major link between integrin and the cytoskeleton. It plays a crucial role in focal adhesion assembly (25, 69) and in integrin-mediated signaling since it is able to recruit focal adhesion kinase (31). Talin is a member of the FERM-containing domain protein family. This 30-kDa domain is a cysteine-rich basic charged globular module. This motif has been reported to bind phosphoinositides and to target proteins to the plasma membrane (42). Several proteins without a FERM domain are known to interact with phosphoinositides. Generally, they associate with phosphoinositides via sequences rich in basic and polar amino acids. It has been speculated that these motifs may facilitate membrane association of these proteins by interacting with the negatively charged head groups of phosphoinositides (70). A consensus sequence for PI4,5P2 binding has been described as (K/R)XXXKX(K/R)(K/R) (71), but other binding sequences exist. The sequence of the head domain of talin is particularly rich in basic amino acids (41), and so talin appears as a good candidate for interacting with phosphoinositides. Indeed, in good agreement with previous reports (50), we have found that talin is able to bind in vitro PI, PIP, and PI4,5P2. However, within a phospholipid bilayer, this interaction is restricted to PI4,5P2. These findings fit with the known properties of the FERM-containing protein family.

In vivo, talin seems to bind PI, PIP, and PI4,5P2, but the relative amount of the lipid varies during the time course of cell attachment and spreading (Fig. 2). Although PI and PIP are the major lipid species bound to talin when HeLa cells are just detached from the matrix, PI4,5P2 is found mainly after 10 min of spreading on fibronectin. Similarly, PIP and PI4,5P2 have also been described to be associated with talin in activated but not on resting platelets (72). This result is consistent with the fact that PI4,5P2 synthesis from PIP is dependent on cell adhesion to the extracellular matrix (2). However, during cell spreading talin associates temporarily with PI4,5P2 which suggests that this phosphoinositide regulates the function of talin during cell adhesion. Surprisingly, no lipids are associated with talin on fully spread cells. This could be due to the action of phospholipases or inositol phosphatases that allow the regulation of phosphoinositide levels. Consistent with this hypothesis, it has been shown that synaptojanin, an inositol polyphosphate-5-phosphatase, can hydrolyze the PI4,5P2 bound to actin regulatory protein such as profilin, cofilin, and alpha -actinin (73).

Knowing that phosphoinositide binding can control protein/protein interactions and biological activities, we have studied the effect of phosphoinositides on talin/integrin interaction by an in vitro binding assay. Indeed, as already reported by many other groups, a peptide corresponding to the cytosolic domain of the beta 1 cytosolic tail was able to bind efficiently talin. However, PIP and PI4,5P2 greatly enhanced this interaction (Fig. 4). Furthermore, with whole integrin receptors affinity-purified on fibronectin or on specific antibodies, efficient binding of talin depends on both integrin occupancy by ligand (40, 43) and the presence of PIP or PI4,5P2 (Fig. 5). It has been reported previously that ligand binding to the integrin alpha IIbbeta 3 induces further conformational changes in the extracellular domain of the receptor, resulting in the exposure of neoantigenic sites termed ligand-induced binding sites. The more recent idea is that such a conformational change is transduced through the cell membrane to the cytoplasmic domain of the receptor, thereby exposing cytoplasmic ligand-induced binding site epitopes for the accumulation of potential intracellular cytoskeletal proteins. These results could suggest that the extracellular ligand-binding site and the cytoplasmic ligand-induced binding site epitopes on integrins are conformationally and functionally coupled. Furthermore, recent data have revealed that the interaction between the globular head domain of talin and the beta 3 cytosolic tail occurs in the membrane-proximal region of the integrin cytoplasmic domain (74). Taken together, these results suggest that the talin-binding site close to the membrane proximal region of the integrin cytoplasmic domain is dynamically regulated. Indeed, as is the case for other phosphoinositide-binding proteins, the association of talin with PI4,5P2 resulted in a conformational change of the protein that allowed an increase in proteolytic sensitivity that may correspond to a more open form of the protein. Finally, our results add evidence that talin is itself rendered competent for binding integrins following its interaction with phosphoinositides. Such interaction may stabilize the activated conformational state since it has been shown that overexpression of the talin head increases the proportion of alpha IIbbeta 3 in its activated state (33).

Altogether the data presented here suggest an attractive model in which lipid metabolism may play a major role in the sequential assembly of focal adhesions by interacting directly with some components of these structures. When cells are detached from the extracellular matrix, talin is not in the vicinity of the plasma membrane, is mainly bound to PIP, and could be competent for immediate binding to the integrin provided that the receptors are allowed to interact with their extracellular ligand. However, since talin interacts with the integrin at the membrane level, efficient interaction might depend only on PI4,5P2, as suggested by cosedimentation experiments carried out with liposomes. Ten minutes after initial plating on fibronectin, a large amount of PI4,5P2 is synthesized due to the adhesion-dependent activation of PIP 5-kinase. Binding of PI4,5P2 on talin keeps this protein in a conformation allowing its interaction with integrins and leads to the break of vinculin head to tail interaction and its subsequent binding to talin. However, high level of PI4,5P2 prevents additional actin binding to vinculin (24). Finally, in order to allow PI4,5P2-bound vinculin to bind actin, the PI4,5P2 should be hydrolyzed or displaced from vinculin (24). By knowing that some inositol 5-phosphatases can hydrolyze the PI4,5P2 bound to cytoskeletal proteins (73), it is possible that this hydrolysis may promote vinculin/actin interaction. Finally, after the organization of stress fibers, fully functional focal adhesions could be formed.

Our data with transient transfection experiments with PH domains are in good agreement with this model, since they showed that PI4,5P2 is necessary for the maintenance of talin in focal adhesions. This could be explained by the observation that PI4,5P2 promotes a high affinity interaction between talin and engaged integrins. PI4,5P2 then recruits and opens talin to expose its binding sites for integrins, thereby contributing to the assembly of focal adhesions. This is also consistent with the fact that the phosphoinositides have no further effect on the binding of the talin head to the cytoplasmic domain of integrin. The transient interaction of PI4,5P2 with talin suggests that the lipids are required only in the early stages of assembly of the new focal adhesions. However, the delocalization of talin from focal adhesions (Fig. 8) does not reflect any discrepancy with this hypothesis. Indeed, PI4,5P2 sequestrated by the PH domain is no longer available for the change of talin conformation and its incorporation in focal adhesions. Contrary to what happens in in vitro binding experiments, focal adhesions are dynamic structures, subjected to a fast turn over. Therefore, even if phosphoinositides are not required to maintain the stability of fully assembled structures, the blockade of talin/integrin interaction results in the progressive disappearance of talin from these structures. On the other hand, vinculin is not displaced from focal adhesions when PI4,5P2 is sequestrated, which agrees with the fact that PI4,5P2 should be displaced from vinculin for allowing its interaction with actin (24). The remaining question is know how vinculin can be attached to integrin without any talin and whether another factor is able to bind vinculin to integrin. This restructuring of focal adhesions indicates that vinculin plays an important role in the stabilization and maintenance of actin stress fibers. Conversely, talin is one of the first associated components in focal adhesion assembly (26). Examination of the localization of focal adhesion proteins (Figs. 8 and 9) indicates that PI4,5P2 sequestration actually induces a restructuring of the focal adhesion plaque rather than a complete dispersal of the complex. However, vinculin remained clustered after 16 h of PH/PLCdelta transfection indicating a hierarchy between different focal adhesion proteins that allow different integrin responses.

Finally, the latter results open very exciting research fields, since they suggest that factors that regulate the assembly and disassembly of focal adhesions are probably different: when PI4,5P2 is sequestrated, talin is one of the first proteins to be excluded, whereas vinculin, integrins, and actin remain associated in focal adhesions for a longer period. This consequence could reflect the availability of focal adhesions to redistribute focal contact proteins and adds another piece of proof of the dynamics of focal contacts.

    ACKNOWLEDGEMENTS

We are very grateful to Lucile Vignoud for expert assistance in plasmid construction, production, and purification of His6-tagged fusion beta 1 cytoplasmic domain. We thank Dr. B. Payrastre for helpful comments and critical review. We also thank Geneviève Tavernier and Brigitte Peyrusse for technical assistance.

    FOOTNOTES

* This work was supported in part by CNRS, a grant from the Ligue Nationale Contre le Cancer, the ARC, the Association Espoir, and the Fondation pour la Recherche Médicale.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by a fellowship from the Ministère de la Recherche et de l'Enseignement Supérieur and the Ligue Nationale Contre le Cancer.

Published, JBC Papers in Press, March 28, 2001, DOI 10.1074/jbc.M102373200

    ABBREVIATIONS

The abbreviations used are: PI4, 5P2, phosphatidylinositol 4,5-bisphosphate; PC, phosphatidylcholine; PI, phosphatidylinositol; PIP, phosphatidylinositol 4-monophosphate; FERM proteins, band 4.1/Ezrin/Radixin/Moesin; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PH, pleckstrin homology; PLCdelta , phospholipase Cdelta ; TBS, Tris-buffered saline; GFP, green fluorescent protein; alpha -MEM, minimum essential medium with alpha  modification; ELISA, enzyme-linked immunosorbent assay.

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ABSTRACT
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RESULTS
DISCUSSION
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