Correspondence to: Fedor Berditchevski, CRC Institute for Cancer Studies, The University of Birmingham, Egdbaston, Birmingham, B15 2TA, United Kingdom., f.berditchevski{at}bham.ac.uk (E-mail), 44-121-414 7458 (phone), 44-121-414 4486 (fax)
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Abstract |
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Tetraspanins (or proteins from the transmembrane 4 superfamily, TM4SF) form membrane complexes with integrin receptors and are implicated in integrin-mediated cell migration. Here we characterized cellular localization, structural composition, and signaling properties of 3ß1TM4SF adhesion complexes. Double-immunofluorescence staining showed that various TM4SF proteins, including CD9, CD63, CD81, CD82, and CD151 are colocalized within dot-like structures that are particularly abundant at the cell periphery. Differential extraction in conjunction with chemical cross-linking indicated that the cell surface fraction of
3ß1TM4SF protein complexes may not be directly linked to the cytoskeleton. However, in cells treated with cytochalasin B
3ß1TM4SF protein complexes are relocated into intracellular vesicles suggesting that actin cytoskeleton plays an important role in the distribution of tetraspanins into adhesion structures. Talin and MARCKS are partially codistributed with TM4SF proteins, whereas vinculin is not detected within the tetraspanin-containing adhesion structures. Attachment of serum-starved cells to the immobilized anti-TM4SF mAbs induced dephosphorylation of focal adhesion kinase (FAK). On the other hand, clustering of tetraspanins in cells attached to collagen enhanced tyrosine phosphorylation of FAK. Furthermore, ectopic expression of CD9 in fibrosarcoma cells affected adhesion-induced tyrosine phosphorylation of FAK, that correlated with the reorganization of the cortical actin cytoskeleton. These results show that tetraspanins can modulate integrin signaling, and point to a mechanism by which TM4SF proteins regulate cell motility.
Key Words: integrin, tetraspanin, adhesion complexes, signaling, cytoskeleton
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Introduction |
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TETRASPANINS, or tetraspan proteins, are a large family (transmembrane 4 superfamily [TM4SF]1) of ubiquitously expressed membrane proteins that are implicated in a number of basic biological phenomena, including cell proliferation, cell migration, and tumor cell invasion (
Notably, TM4SF proteins form membrane complexes with adhesion receptors from the integrin family (
In this study, we have characterized the properties of integrinTM4SF protein complexes, particularly, in relation to different types of cellECM adhesion structures (e.g., focal adhesions, focal contacts, and point contacts). Our data indicate that TM4SF proteins are mostly excluded from the vinculin-containing adhesion complexes (both focal adhesions and focal complexes), but are coclustered with 3ß1 integrin within peripheral adhesion structures, some of which contain talin and MARCKS. Furthermore, we have demonstrated that TM4SF proteins may contribute to integrin-mediated signaling.
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Materials and Methods |
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Cell Lines
Human breast carcinoma cell line, MDA-MB-231, was purchased from American Type Culture Collection and maintained in L-15 medium Leibovitz supplemented with 15% fetal calf serum. Human fibrosarcoma cells, HT1080, were grown in DME supplemented with 10% fetal calf serum. HT1080/zeo cells were generated by transfection of pZeoSV (Invitrogen) into HT1080 cells. HT1080-CD9 cells were generated as follows: HindIII-XbaI fragment of CD9 cDNA (
Antibodies
The anti-TM4SF mAbs used were C9-BB, anti-CD9 (2 (
3 (
5 (
6 (
3 integrin subunit was a generous gift from Dr. F. Watt (ICRF, London, UK). Monoclonal anti-MARCKS antibody, 2F12, was from Dr. P. Blackshear (National Institute of Environmental Health Sciences, Durham, NC). Anti-CD9 mAb, BU16, were purchased from The Binding Site; anti-CD81 mAb, JS64, were purchased from Serotech. Anti-CD44 mAb, clone F10-44-2, was purchased from Novosastra Laboratories Ltd. Anti-vinculin mAb, clone hVIN-1, and anti-talin mAb, clone 8d6, were from Sigma. Other antibodies used were W6/32, antiMHC class I (
Adhesion Assay
A standard static adhesion assay (3035 min) was carried out as previously described (
Immunoblotting
In the experiments studying the solubility of membrane proteins, cells were plated on ECM-coated dishes for 1.52 h in serum-free DME. Membrane proteins were solubilized for 10 min at 4°C into 0.2% Triton X-100/PBS (or for 20 min into 1% Tween 20/PBS) supplemented with a cocktail of protease inhibitors, and then insoluble material was precipitated at 12,000 rpm for 10 min at 4°C. Tween and Triton lysates were appropriately supplemented with 4x Laemmli buffer and treated for 5 min at 95°C. Detergent-insoluble proteins were reextracted into Laemmli loading buffer at 95°C for 10 min. Proteins were separated in 10% SDS-PAGE, and, after transferring to nitrocellulose membrane, were probed with appropriate primary Ab. Protein bands were visualized with HRPO-conjugated goat antirabbit or goat antimouse Ab (both from Sigma) using ECL reagent (Amersham).
In studies of FAK tyrosine phosphorylation, cells were plated on bacteriological dishes precoated with either ECM proteins (collagen, laminin) at 10 µg/ml or mAbs at 5 µg/ml for 1 h. In some experiments collagen (2.5 µg/ml) was coimmobilized on bacteriological dishes together with purified goat antimouse IgG Ab (10 µg/ml) overnight at 4°C. The dishes were subsequently blocked with heat-denatured BSA for 2 h at 37°C and then incubated with an appropriate mAb (15 µg/ml) for 3 h at 37°C. Cells were solubilized in 1% Triton X-100 lysis buffer for 1 h at 4°C, and FAK was immunoprecipitated by using polyclonal Ab immobilized on protein Aagarose beads. Immunoprecipitated material was eluted from the beads into Laemmli loading buffer and resolved in 10% SDS-PAGE. After transferring to nitrocellulose membrane, proteins were probed with Ab as described above.
Immunofluorescent Staining
For immunofluorescence analyzes cells were plated on glass coverslips coated with ECM ligands for 12 h. When spread, cells were fixed for 710 min with 2% paraformaldehyde in PBS, containing 5% sucrose and 2 mM MgCl2, and then treated with 1% Brij 98 in PBS for 2 min. In some experiments, fixed cells were permeabilized with 0.5% CHAPS in PBS for 2 min, or 0.25% Triton X-100 for 1 min. Coverslips were blocked for 1 h with 20% heat-inactivated normal goat serum, HI-NGS, in PBS. Cells were then stained with primary mAbs diluted in 20% HI-NGS in PBS. Staining was subsequently visualized with FITC-conjugated goat antimouse serum (Sigma Chemical Co.) before the coverslips were mounted with FluorSave (Calbiochem-Novabiochem), and immunofluorescence was examined using a Zeiss Axioscop. Serial Z-sections (0.2 µM) of stained cells captured with Coolview CCD camera (Photonic Sciences) were digitally saved by using Biovision software package (Bio-Rad Laboratories). The images were further processed by using a digital deconvolution module of the OpenLab software package (Improvision). For colocalization experiments, paraformaldehyde-fixed and permeabilized cells were first incubated with a combination of mouse mAbs, and then visualized with a combination of isotype specific goat antimouse sera coupled to FITC (anti-IgG2A; anti-IgG2B) and to Rhodamine (anti-IgG1).
Immunoelectronmicroscopy
Cells were plated on 20-mm Termoplax coverslips precoated with laminin-5containing ECM for 1 h in serum-free media. Paraformaldehyde-treated cells were stained with primary mAb as described above, and subsequently incubated with rabbit antimouse IgG conjugated with 10-nm gold particles (Amersham International). After washing, the stained cells were additionally fixed with 0.2% glutaraldehyde for 2 h at room temperature. Samples were dehydrated and embedded in Lowicryl HM20 resin. Ultrathin sections were taken from the embedded sample and collected on nickel grids. The sections were stained in 30% uranyl acetate in 100% methanol for 5 min, washed in water, and dried. The sections were then analyzed using Jeol 1200 EX transmission electron microscope.
Immunoprecipitation
Cells (23 x 106) were plated on ECM coated surface for 2 h in serum-free DMEM. Cells were washed 3 times with PBS and lysed in immunoprecipitation buffer (1% Brij 98 in PBS, containing 2 mM MgCl2, 2 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin) for 1 h at 4°C. Insoluble materials were pelleted at 12,000 rpm for 10 min, and the cell lysates were precleared by incubation with agarose beads, conjugated with goat antimouse antibodies for 30 min at 4°C (Sigma). Immune complexes were collected onto the agarose beads that were prebound with appropriate mAb, followed by four washes with the immunoprecipitation buffer. In some experiments Brij 98 in the immunoprecipitation buffer was substituted for Brij 96, CHAPS, or Triton X-100 (all used at final concentration 1%). Immune complexes were eluted from beads with Laemmli sample buffer and resolved by 812% SDS-PAGE. In cross-linking experiments, spread MDA-MB-231 cells were washed with a cross-linking buffer (2 mM MgCl2, 5 mM KCl, 5 mM glucose, 140 mM NaCl, 20 mM Hepes, pH 7.4), and subsequently treated with 0.4 mM 3'3'-Dithiobis(sulfosuccinimidyl)propionate (Pierce & Warriner) in a cross-linking buffer for 20 min at room temperature. After additional washes with PBS, the cells were scraped into a lysis buffer containing 1% Tween 20, and immunoprecipitation was carried out as described above, except that immune complexes collected on the beads were washed with a lysis buffer supplemented with 0.1% SDS.
Flow Cytometry
Cells were incubated with saturating concentrations of primary mouse mAbs for 45 min at 4°C, washed twice, and then labeled with fluorescein isothiocyanate (FITC)-conjugated goat antimouse immunoglobulin. Stained cells were analyzed on a FACScan® (Becton Dickinson).
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Results |
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TM4SF Proteins Are Colocalized within Adhesion Complexes
We have previously found that in human fibrosarcoma cells, HT1080, two TM4SF proteins, CD63 and CD81, are localized at the cell periphery and on intracellular vesicles (
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To examine whether different TM4SF proteins are colocalized with each other within adhesion structures we carried out double-labeling immunofluorescent experiments. Figure 3 shows staining of MDA-MB-231 cells with three combinations of anti-TM4SF mAbs, CD9-CD63, CD81-CD151, and CD82-CD151. In these, and in the experiments where other combinations of anti-TM4SF mAbs were used, we observed notable colocalization of the proteins within dot-like adhesion complexes that was particularly prominent at the cell periphery (Figure 3C, Figure F, and Figure I). In another set of experiments we have demonstrated that 3ß1 integrin is colocalized with TM4SF proteins within these adhesion structures (Figure 3, JL). Similarly, tetraspanins were codistributed with one another and with
3ß1 integrin in cells plated on collagen and fibronectin (data not shown). Abundance of
3ß1TM4SF protein complexes at the cell periphery was further confirmed by immunoelectron microscopy (Figure 4A and Figure B). Colocalization of TM4SF proteins with
3ß1 integrin agrees with the immunoprecipitation data that have shown that in MDA-MB-231 cells this integrin is associated with four different tetraspanins, including CD9, CD81, CD82, and CD151 (Figure 5 A). In contrast, mAbs recognizing
2ß1 or
5ß1 integrins did not coimmunoprecipitated these tetraspanins (Figure 5 A).
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Previous reports suggested that TM4SF proteins may affect integrin-mediated cell adhesion (3- and
6-integrin subunits had only a partial inhibitory effect on adhesion MDA-MB-231 cells to laminin-5containing matrix, suggesting that cellular interactions with the substrate involve other integrin receptors (e.g.,
1ß1 or
2ß1 integrins). Collectively, these experiments indicate that
3ß1tetraspanin peripheral adhesion complexes are not directly involved in mediating strong cellECM attachment that is tested in static adhesion assays.
TM4SF Proteins Are Absent from Focal Adhesions and Point Contacts
To analyze the composition of TM4SF-containing adhesion complexes further, we studied colocalization of tetraspanins with a number of cytoplasmic proteins, which are typically associated with various adhesion structures. In these experiments cells were labeled with a cocktail of anti-TM4SF mAbs to visualize all various TM4SF-containing protein complexes. Initial experiments were carried out using serum-starved MDA-MB-231 cells. Figure 6 illustrates that under serum-free conditions vinculin was found in dot-like adhesion structures at the peripheral locations as well as in rear focal adhesions (Figure 6 B). Notably, only a few of the vinculin-containing punctate adhesion structures and none of the focal adhesions included TM4SF proteins (Figure 6A and Figure C). Because of its established role in the assembly of various types of adhesion complexes we investigated whether fetal calf serum can facilitate colocalization of tetraspanins with vinculin. As expected, in the presence of fetal calf serum the number of focal adhesions was increased, but the treatment failed to direct TM4SF proteins into vinculin-containing adhesion structures (Figure 6, DF). Similar results were obtained when we examined the codistribution of tetraspanins with paxillin and VASP (data not shown). In contrast, under the serum-free conditions a substantial number of tetraspanin-containing peripheral adhesion structures included talin (Figure 6, GI). More strikingly, another actin binding protein, MARCKS, was colocalized with tetraspanins in the peripheral and in some of the centrally located adhesion complexes (Figure 7, AC).
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Finally, we investigated whether integrinTM4SF adhesion complexes contain components of clathrin coated structures, that are known to be associated with point contacts (
Peripheral Localization of TM4SF Proteins Is Dependent on Intact Actin Cytoskeleton
Assembly of various types of adhesion complexes is intimately linked to the reorganization of cytoskeleton (3ß1TM4SF protein complexes in MDA-MB-231 cells treated with cytochalasin B or nocodazole, drugs that induce destabilization of actin filaments and depolymerization of microtubules, respectively. Cytochalasin B induced the collapse of lamellipodia in most cells with few residual protrusions left extending from the rounded cell bodies. Figure 8 illustrates that in cytochalasin-treated cells both
3ß1 integrin and TM4SF proteins were found predominantly on intracellular vesicles (Figure 8, AC). In contrast, in cells treated with nocodazole,
3ß1TM4SF protein complexes were not only retained at the cell periphery, but, in some cells, were redistributed into large peripheral clusters (Figure 8, DF). Immunoprecipitation experiments and subsequent densitometric measurements showed that the amount of
3ß1 integrin coimmunoprecipitated with TM4SF proteins from both nocodazole- and cytochalasin-treated cells was only slightly decreased (by 45 and 20%, respectively) comparing to the control sample (Figure 9, lanes 46). Thus, we have concluded that both actin cytoskeleton and microtubules may affect cellular distribution of
3ß1TM4SF protein complexes.
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The 3ß1TM4SF Protein Complexes Are Not Directly Linked to Cytoskeleton
During the course of immunofluorescence staining we noticed that in cells that were permeabilized with detergents other than Brij 98 (e.g., Triton X-100 or CHAPS), TM4SF proteins and 3ß1 integrin could no longer be detected at the peripheral locations (not shown). These results suggested to us that the linkage between
3ß1tetraspanin protein complexes and the cytoskeleton is weak. To test this hypothesis we studied the solubility of TM4SF proteins in Triton X-100. As shown in Figure 10 A, TM4SF proteins and
3ß1 integrin were detected mostly in Triton-soluble fraction. Importantly, similar results were obtained regardless of whether MDA-MB-231 cells were plated on laminin-5containing matrix or collagen I, suggesting that direct ligation of
3ß1 integrin by its most avid ligand (laminin-5) does not affect solubility of
3ß1tetraspanin complexes. In contrast, ~3035% of CD44 was insoluble under the same experimental conditions. As not all
3ß1 and TM4SF proteins presented on the cell surface are engaged in interactions with each other (
3ß1 and tetraspanins between soluble and insoluble fractions thus permitting to assess directly association of the complexes with the cytoskeleton. Of various detergent tested for this purpose, including CHAPS, Brij 96, Brij 98, and Tween 20, we found that the latter gives the most reproducible results when 2050% of the total amounts of tetraspanins and
3ß1 integrin could be solubilized from the membranes of MDA-MB-231 cells (Figure 10 B). To assess the solubility of the cell surface fraction of
3ß1TM4SF protein complexes directly, we pretreated intact MDA-MB-231 cells with a membrane-impermeable chemical cross-linker before solubilization with Tween. Subsequently, we purified the complexes from the Tween-soluble fraction and from the pellet (that was reextracted with Tween containing 0.1% SDS) by immunoprecipitation using a mixture of anti-tetraspanin mAbs, and compared the amounts of
3ß1 integrin present in the immunoprecipitates. Importantly, all immunoprecipitation steps were carried out in the presence of 0.1% SDS to dissociate the intracellular complexes, which were inaccessible to the action of the cross-linker. As shown in Figure 10 C,
3ß1 integrin was almost exclusively detected in the Tween-soluble fraction. Together these results provide a strong support for the idea that the linkage of
3ß1TM4SF protein complexes to the cytoskeleton is weak.
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A Role of IntegrinTM4SF Protein Complexes in Signaling
Three approaches were used to study a contribution of TM4SF proteins in integrin-mediated signaling. First, we examined phosphorylation of focal adhesion kinase induced by adhesion of MDA-MB-231 cells to immobilized anti-integrin and anti-TM4SF mAbs. After 1 h of incubation, serum-starved MDA-MB-231 cells appeared well spread on a control ECM substrate (collagen) and anti-integrin mAbs. In contrast, most cells plated on a mixture of anti-TM4SF or antiMHC class I mAbs were rounded and developed only short projections. As expected, in cells attached to collagen and anti-integrin mAbs the level of tyrosine phosphorylation of FAK was 815 times higher of that in cells kept in suspension (Figure 11 A, top, lanes 14). Surprisingly, tyrosine phosphorylation of FAK in cells attached to a mixture of anti-TM4SF mAbs (lane 5) was reduced by 2.5-fold comparing to suspended cells. Importantly, we found that tyrosine phosphorylation of FAK was essentially identical in suspended cells and in cells attached to antiMHC class I mAb (Figure 11 A, top, lane 6). In additional experiments we found that the levels of tyrosine phosphorylation of FAK were also lower in the cells plated on the separately immobilized anti-TM4SF mAbs (Figure 11 B, top) with a stronger effect observed for the anti-CD63, -CD82, and -CD151 Ab. Control experiments confirmed that similar amounts of FAK were immunoprecipitated in each case (Figure 11a and Figure b, bottom panels). Second, we investigated the effect of the clustering of tetraspanins with mAbs on tyrosine phosphorylation of FAK in cells plated on ECM ligand. To this end, the MDA-MB-231 cells were plated on a mixed substrate that included collagen and anti-tetraspanin mAbs. To standardize the amounts of the immobilized mAb in each case, the immobilization of the substrates was carried out in two steps. First, the bacteriological dishes were incubated with a solution containing collagen and purified goat antimouse IgG Ab (see Material and Methods for details). Second, the anti-TM4SF or antiMHC class I mAbs were captured on the dishes after an additional incubation at 37°C. As illustrated in Figure 11 C, all tested anti-TM4SF mAbs (but not a control antiMHC class I mAb) potentiated collagen-induced tyrosine phosphorylation of FAK.
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Finally, we investigated how the ectopic expression of TM4SF proteins affects ECM-stimulated tyrosine phosphorylation of FAK. For these experiments we used a newly developed pair of cell lines HT1080/zeo and HT1080/CD9. When analyzed by flow cytometry, expression levels of ß1 integrins on HT1080/zeo and HT1080/CD9 cells were comparable (Table 1). Furthermore, HT1080/zeo and HT1080/CD9 cells showed no differences in their ability to attach and spread on various ECM substrates, including collagen, fibronectin, and laminins (Figure 12). Tyrosine phosphorylation of FAK was assessed in cells spread on collagen type I and laminin-1. As seen in Figure 13 A, adhesion of HT1080/zeo cells to collagen caused the increase of FAK tyrosine phosphorylation by ~17-fold (lanes 1 and 3), whereas in HT1080/CD9 cells the increase was somewhat less dramatic (~8-fold, lanes 2 and 4). On the contrary, when attached to laminin-1 induction of tyrosine phosphorylation of FAK was stronger for HT080/CD9 cells (~5.5- versus ~3.5-fold for HT1080/zeo cells; Figure 13 A, lanes 5 and 6). Time-course experiments with the cells plated on laminin have demonstrated that initial increase in tyrosine phosphorylation of FAK (20 min) was comparable in both cell lines (Figure 13 B, lanes 2 and 6). However, by 2 h the differences became apparent: in HT1080/zeo cells the level of FAK phosphorylation has gradually decreased whereas no significant changes were observed in HT1080/CD9 cells (Figure 13 B, lanes 4 and 8). Taken together, these results imply that signaling through integrinTM4SF protein complexes may contribute to adhesion-dependent activation of FAK.
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IntegrinTM4SF Protein Complexes Are Involved in Reorganization of Actin Cytoskeleton
Activation of FAK is known to be dependent on the organization of actin cytoskeleton. Thus, we examined whether observed differences in phosphorylation of FAK in HT1080/zeo and HT1080/CD9 cells were related to the distribution of filamentous actin. No significant differences were found between the cell lines when we quantified the F-actin contents in the cells plated on collagen or laminin (not shown). Surprisingly, immunofluorescence staining with rhodamine-conjugated phalloidin revealed that in 6070% of HT1080/zeo cells plated on collagen, actin filaments at the cortical areas were more abundant than in HT1080/CD9 cells (Figure 14A and Figure B), and opposite results were obtained when the cells were plated on laminin (Figure 14C and Figure D). Thus, although tetraspanins do not seem to have a role in integrin-induced actin polymerization, their function may be related to adhesion-dependent reorganization of actin filaments in the cortical areas.
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Discussion |
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Migrating cells interact with the extracellular matrix using various types of adhesion structures, including initial adhesion contacts that the cell makes at the periphery of extending protrusions, point contacts, focal complexes, and focal adhesions. Focal complexes and focal adhesions, that represent anchoring sites for actin filaments, confer strong cellECM interactions and, therefore, may play an important role in guiding cellular protrusions and generating traction forces. On the other hand, adhesive interactions that take place at the edge of cellular extensions are more dynamic: by using real-time video microscopy it was observed that the frontal edge of lamellipodial protrusions and tips of filopodia frequently undergo a few cycles of detachment and reattachment before the protrusions are firmly fixed on the substrate (3ß1 integrins at the most distal parts of lamellipodial and filopodial extensions, an appropriate location for proteins that are involved in attachment-detachment cycles. Second, the link between
3ß1TM4SF protein complexes and cytoskeleton appears to be weak, suggesting that adhesive interactions mediated by the complexes are not constrained by the cytoskeletal proteins. Third, depending on receptor occupancy, integrinTM4SF protein complexes may deliver functionally opposite signals, causing rapid reorganization of actin cytoskeleton at the periphery of cellular extensions, thus supporting dynamic interactions between the protrusions and a substrate.
Most cells simultaneously express more than one member of TM4SF and at least one tetraspanin-associated integrin (
One of the main objectives of this study was to relate TM4SF-containing adhesion complexes to other types of adhesion structures (e.g., focal contacts/focal adhesions, focal complexes, and point contacts). In the previous report, we have found that CD63 and CD81 are clustered in punctate adhesion structures that are morphologically distinct from focal contacts (
The cytoskeleton (both actin filaments and microtubules) appears to play an important role in the cellular distribution of the integrinTM4SF protein complexes. One possibility is that the actin-based cell cortex is acting as a physical barrier to endocytosis of the complexes, a process that, in turn, may be dependent on the microtubular cytoskeleton. This would explain the apparent incompatibility of tetraspanins with focal adhesions: destabilization of actin filaments at the cell cortex that occurs while cell extends its protrusions will make integrinTM4SF protein complexes more susceptible to internalization and therefore preclude their occurrence in focal adhesions. In this regard, colocalization of tetraspanins with MARCKS, a protein that is thought to promote actin dynamics at the cell periphery during cell spreading (
Our results clearly demonstrate direct involvement of TM4SF protein complexes in integrin-dependent signaling. Tyrosine phosphorylation of FAK is a well established hallmark of outside-in integrin signaling that depends on the rearrangement of the actin cytoskeleton. Given the fact that the level of FAK tyrosine phosphorylation can be affected by tetraspanins (either via antibody clustering or by comparing adhesion-dependent responses using cell transfectants), we propose that TM4SF proteins are signaling modulators of integrin-mediated reorganization of the cortical actin cytoskeleton. In principal, ligated integrins can affect the actin cytoskeleton in two ways. First, certain cytoskeletal proteins are constitutively associated with integrin cytoplasmic tails, thus providing docking sites for the attachment of actin filaments. In this regard, the accumulation of talin and MARCKS (both actin-binding proteins) within integrinTM4SF adhesion complexes may be sufficient for tetraspanins to sterically transmit their modulatory effect to the actin cytoskeleton. Although possible, the idea that integrinTM4SF protein complexes play only a mechano-scaffolding role in the spatial organization of actin filaments is somewhat confronting the fact that a link between the complexes and the cytoskeleton is weak. On the other hand, the signaling scenario proposes that integrins may affect actin cytoskeleton by triggering distinct signaling pathways (for example, through activation of protein or/and phosphoinositide kinases) (3ß1 integrin (Berditchevski, F., unpublished data).
Interestingly, our data indicate that the modulatory impact from tetraspanins can be both negative and positive, perhaps depending on whether or not tetraspanin-associated integrins are engaged in ligand binding, and on a nature of the ligand. Indeed, dephosphorylation of FAK observed upon cell detachment suggests that disengaged and ligand bound integrins trigger opposite biochemical signals. Thus, it is conceivable that when integrins are unoccupied, additional clustering with anti-TM4SF mAbs may enhance negative signals emanating from the tetraspanin-associated integrins. Conversely, additional clustering occurring in cells plated on ECM ligands (e.g., when integrins are engaged) can potentiate positive signaling outcome. The results describing an opposite, ligand-dependent effect of CD9 on adhesion-mediated tyrosine phosphorylation of FAK in HT1080 cells, are particularly interesting. Again, it is possible that in cells plated on collagen, conformations assumed by 6ß1 and
3ß1, two tetraspanin-binding integrins in HT1080 cells, favor signaling pathways that counteract ligand-dependent phosphorylation of FAK mediated by the
2ß1 integrin, a principal collagen receptor in HT1080 cells. On the other hand, when cells are plated on laminin, CD9 enhances positive (e.g., leading to phosphorylation of FAK) signaling via ligand-bound
6ß1 integrin. Although exact mechanisms remains to be uncovered, the observed modulatory effect of the tetraspanins on adhesion-dependent signaling may involve the enzymes that are associated with integrinTM4SF protein complexes (e.g., protein kinases or phosphoinositidylinositol 4-kinase (
-actinin, and MARCKS, cytoskeletal proteins that are themselves either directly bound or localized in a close proximity to integrins (
Various signaling pathways link actin cytoskeleton reorganization with phosphorylation of FAK, including trans-phosphorylation caused by integrin clustering, phosphorylation by activated tyrosine kinases of src family, or through the involvement of cytosolic protein tyrosine phosphatases (
Whether influenced by TM4SF proteins directly (through the actin cytoskeleton rearrangement) or indirectly (via activation of other signaling pathways), the level of tyrosine phosphorylation of FAK is a critical factor that determines a molecular architecture of adhesion complexes, their signaling capacities, and, ultimately, their role in cell migration (
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Acknowledgements |
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We thank all our colleagues who provided us with the reagents used in this study. We also thank L. Tompkins for invaluable help in preparation of the sections for immunoelectron microscopy and Dr. T. Sugiura, M. Griffiths, and E. Gilbert for critical review of the manuscript.
Work was supported by the Cancer Research Campaign grant SP2369/0101 (to F. Berditchevski).
Submitted: September 28, 1998; Revised: May 5, 1999; Accepted: June 2, 1999.
1.used in this paper: FAK, focal adhesion kinase; TM4SF, transmembrane 4 superfamily
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References |
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