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INTRODUCTION |
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
3
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
-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
-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
-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
1 and
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
1
integrin. Finally our data strongly suggest a possible role of
phosphoinositides in the sequential assembly of focal adhesions.
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EXPERIMENTAL PROCEDURES |
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. [
-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
1 integrin was from Immunotech. Rabbit anti-talin and anti-
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
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 PLC
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 PLC
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'; PLC
5'-AAAAAACTCGAGCAATGGACTCGGGCCGGGACTTCCTG-3'; and PLC
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
1 integrin
cytoplasmic domain was produced in the BLR(DE3) pLysS Escherichia
coli strain containing the vector pET19b-Cyto
1.
This construct allows the production of the fragment 752-798 of the
1 integrin cytoplasmic domain. This peptide was
recognized by a polyclonal antibody raised against a synthetic
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
modification (
-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 PLC
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
-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 [
-32P]phosphate in
-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
1
Peptides--
A 47-amino acid peptide corresponding to the cytoplasmic
domain of
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
1 peptides coated in the wells was
estimated by ELISA with a polyclonal antibody directed against the
cytoplasmic domain of
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
-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
1 integrin was estimated by ELISA with a
polyclonal antibody directed against the cytoplasmic domain of
1 integrin.
 |
RESULTS |
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.
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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.
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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.
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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
and
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
1,
3, and
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
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
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
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
1
integrin (Fig. 4A, lanes 6 and 7),
whereas it has no effect on the coating of
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
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 1 cytoplasmic
domain. A, microtiter plate was coated with 10 µg of
the peptide corresponding to the 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 1 integrin has been performed to check that
equal amounts of 1 peptides have been coated in each
well. The amount of 1 peptides does not change
regardless of the phosphoinositides added. The figure illustrates
one representative experiment of four performed with similar
results.
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Talin was also reported to interact with both the cytoplasmic domains
of
and
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
1
integrin in the wells (Fig. 5A').

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Fig. 5.
Phosphoinositides modulate the interaction
between talin and 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 1 integrin
has been performed to check that equal amounts of 1
integrin have been retained in each well (A', B', and
C'). The amount of 1 peptides does not
change regardless of the phosphoinositides added. The figure
illustrates one representative experiment of four performed with
similar results.
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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-
1
antibody-coated wells. Two different antibodies were used as follows:
either the monoclonal antibody K20 that recognizes an extracellular
epitope of the
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
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
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
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
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
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 1 integrin
has been performed to check that equal amounts of 1
peptides have been coated in each well. The amount of
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
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.
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Binding of PI4,5P2 on Talin Increases Its Affinity for
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
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
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.
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In order to estimate the effect of PI4,5P2 on affinity of
talin for
1 integrin, we performed the previous solid
phase assay using immobilized
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
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/
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 PLC
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
PLC
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(PLC
) 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(PLC
), 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 PLC
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 PLC
or
ARNO. So, when the plasma membrane PI4,5P2 is sequestrated
by GFP-PLC
, 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-PLC /PH domain has
no effect on the distribution of vinculin (B). On the
contrary, overexpression of GFP-PLC /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 PLC . 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 PLC - 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-PLC (black
bars). Results are representative of three separate
experiments.
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DISCUSSION |
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
-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
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
IIb
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
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
IIb
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/PLC
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.