INTRODUCTION

T cells continuously change from a mobile to a sessile state.They leave the blood either to home into lymph nodes or tomigrate into sites of infection (Brown, 1997; Butcher andPicker, 1996). Furthermore, T cell activation as well as effectorfunctions are initiated by intimate contact between T cells andantigen presenting cells (APC) and target cells, respectively(Geiger et al., 1982; Ryser et al., 1982). For T cell maturation,too, an intimate contact with stromal cells is important(Kobayashi et al., 1994). The changes between the mobile andthe sessile state are accompanied by adhesion, flattening andspreading (Reinhold et al., 1999; Rosenman et al., 1993).These processes require at the initial state the engagement ofadhesion molecules (Dustin and Springer, 1989; Kaga et al.,1998), which trigger signal transducing molecules involved inthe reorganization of the cytoskeleton (Shaw and Dustin, 1997;Wülfing and Davis, 1998). The rearranged cytoskeleton maythen serve as a scaffold for cascades of signal transducingmolecules inducing gene activation and transcription(Penninger and Crabtree, 1999).

The cytoskeleton is a highly dynamic structure thatreorganizes when cells respond to extracellular stimuli bydivision and/or changes in shape or activity. The key player forthe cellular shape is the actin cytoskeleton (Howard and Watts,1994). Changes in plasma membrane morphology are due toactin polymerization and rearrangement of the underlyingcortical actin filaments. Signaling molecules involved inpolymerization are tyrosine kinases, small GTPases of the Rho

subfamily and membrane phospholipids (Harder and Simons,1999; Janmey, 1998; Mackay and Hall, 1998). As explored infibroblasts, activation of the Rho subfamily members, Rho, Racand Cdc42, trigger formation of actin stress fibers and focaladhesion complexes (Rho), actin polymerization at the plasmamembrane to produce lamellipodia and membrane ruffles(Rac), filopodial protrusions and microspikes (Cdc42) (Kozmaet al., 1995; Ridley et al., 1992). Target molecules of the Rhosubfamily include WASP and p95vav (Holsinger et al., 1998;Miki et al., 1996), the latter being found exclusively inhematopoietic cells. Notably, the involvement of the actincytoskeleton in the process of T cell activation could beconvincingly demonstrated in vav and WASP deficient T cells,which poorly respond to an antigenic stimulus (Holsinger etal., 1998; Kong et al., 1998; Snapper et al., 1998). The sameaccounts for T cells treated with cytochalasin, an agent whichdisrupts the actin cytoskeleton (Holsinger et al., 1998).

The adhesion molecule CD44 has been implicated in allprocesses associated with changes in T cell morphology.Originally it has been described as an adhesion moleculemediating lymphocyte homing (Jalkanen et al., 1988).Meanwhile it is known that CD44 is also involved in theextravasation of activated lymphocytes (DeGrendele et al.,1997), in lytic effector functions (Galandrini et al., 1994;Matsumoto et al., 1998) as well as in T cell activation.Engagement of CD44 has been described to modulate T cellproliferation and cytokine production (Galluzzo et al., 1995;Huet et al., 1989; Sommer et al., 1995; Toyama-Sorimachiet al., 1995), has been implicated in enhanced binding of

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T cell activation is accompanied by actin-mediated receptorclustering and reorganization of lipid rafts. It has beensuggested that costimulatory molecules might be involvedin these processes. We here provide evidence thatengagement of the adhesion molecule CD44 initiatescytoskeletal rearrangement and membrane reorganizationin T cells.

Cross-linking of CD44 on a T helper line wasaccompanied by adhesion, spreading and actin bundleformation. These processes were energy dependent andrequired an intact actin and microtubule system. Theyinvolved the small GTPase Rac as evidenced by the absenceof spreading in cells overexpressing a dominant negativeform of Rac. The CD44 initiated reorganization of the

cytoskeleton was associated with the recruitment of CD44and the associated tyrosine phosphokinases p56lck andp59fyn into glycolipid enriched membrane microdomains(GEM). We interpret the data in the sense that CD44functions as a costimulatory molecule in T cell activationby inducing actin cytoskeletal rearrangements andmembrane protein and lipid reorganization including itsassociation with GEMs. Due to the association of CD44with lck and fyn this colocalization with the TCR allows anabundant provision of these kinases, which are essential toinitiate the TCR signaling cascade.

Key words: Rodent, T lymphocyte, Signal transduction,Cytoskeleton, Adhesion molecule

SUMMARY

Involvement of CD44 in cytoskeleton rearrangementand raft reorganization in T cellsNiko Föger 1, Rachid Marhaba 1 and Margot Zöller 1,2,*1Department of Tumor Progression and Immune Defense, German Cancer Research Center, Heidelberg, Germany2Department of Applied Genetics, University of Karlsruhe, Germany*Author for correspondence (e-mail: m.zoeller@dkfz.de)

Accepted 16 November 2000Journal of Cell Science 114, 1169-1178 © The Company of Biologists Ltd

RESEARCH ARTICLE

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dendritic cells to T cells (St John et al., 1990) and has beensuggested to function as a costimulus in allogeneic andmitogenic T cell responses by binding to the chondroitinsulfate form of the invariant chain (Naujokas et al., 1993).Cross-linking of CD44 has been noted to prolong the Ca2+

influx (Galandrini et al., 1994; Galluzzo et al., 1995; Dianzaniet al., 1999). Furthermore, it has been described that CD44associates with src-family kinases, an interaction which occursin GEMs (Ilangumaran et al., 1998; Oliferenko et al., 1999;Taher et al., 1996). In this context it should be rememberedthat CD44 is known to interact with several cytoskeletalproteins like actin, ankyrin and members of the ERM family(Bourguignon and Jin, 1995; Tsukita et al., 1994). So far,functional importance of the interaction with the cytoskeletononly has been reported for HA binding, which depends on theassociation with ankyrin (Bourguignon et al., 1993) and fortumor cell migration and invasion (Bourguignon et al., 1998).

We recently have described that CD44 exerts costimulatoryfunction under physiological conditions of antigenic T cellactivation (Föger et al., 2000). We could show that cross-linking of CD44 significantly enhances signal transduction viathe TCR/CD3 complex. We interpreted our findings in thesense that the costimulatory function of CD44 relied on itscooperativity with the TCR. Since CD44 was found to beconstitutively associated with p56lck and p59fyn, it becametempting to speculate that it may be the recruitment ofphosphokinases which significantly lowered the threshold forinitiation of signal transduction via the TCR. Here we showthat, indeed, cross-linking of CD44 leads to rearrangement ofthe cytoskeleton, cap formation and the recruitment of theCD44-associated phosphokinases into lipid rafts.

MATERIALS AND METHODS

Cell linesA murine CD4+ T cell clone, IP12-7, specific for influenza virushemagglutinin (HA317-329) has kindly been provided by Dr E.Rajnavolgyi, L. Eotvos University, God, Hungary (Rajnavolgyi et al.,1994). The clone was maintained in RPMI 1640, supplemented with10% FCS and antibiotics. The T helper line D10.G4.1 has beenobtained from the American Type Culture Collection (TIB-224) andhas been maintained according to the suppliers suggestion. Whereindicated IP12-7 cells were transiently transfected with myc epitope-tagged dominant negative mutant forms of RhoA or Rac1 byelectroporation. The eucaryotic expression vector encoding mycepitope tagged dominant negative mutant forms of RhoA (myc-RhoN19) and Rac1 (myc-RacN17) were kindly provided by P.Boquet, INSERM U452, Nice, France (Qui et al., 1995). Aliquots of5×106 IP12-7 cells were resuspended in 400 µl ice-cold serum freeRPMI 1640 and 40 µg of DNA was added. Electroporation wasperformed using an EUROGENTEC electroporation apparatus at asetting of 250 V and 900 µF. Expression of negative mutant formswas controlled by staining with a myc-specific antibody. In oneexperiment lymph node cells of BALB/c mice have been used. T cellswere enriched by passage over a nylon wool column and were keptin culture overnight in the presence of 10 U/ml IL-2.

AntibodiesIM-7 (anti-CD44, rIgG2b) and KM81 (anti-CD44, rIgG2a) have beenobtained from the American Type Culture Collection. 9E10 (anti-myc,mIgG1) has been obtained for the European Collection of Animal CellCultures. Antibodies were purified from culture supernatants bypassage over Protein G-Sepharose. The eluates were concentrated and

filter sterilized. The following monoclonal antibodies were obtainedcommercially: Anti-phopsphotyrosine, anti-fyn, anti-Rac1, anti-RhoA, anti-Cdc42 (all Santa Cruz Biotechnology), FITC-conjugatedgoat anti-rat IgG2b (Bethyl Laboratories), horseradish peroxidase(HRP)-conjugated sheep anti-rat IgG (Amersham Buchler), Cy2- andHRP-conjugated donkey anti-mouse IgG, Texas red- and HRP-conjugated donkey anti-rabbit IgG (Dianova).

Cap formation and spreadingCD44 receptor caps were induced by incubating 1×105 IP12-7 cellsfor 15 minutes at 37°C with IM7 (10 µg/ml). After washing, theprimary antibody was cross-linked using FITC-conjugated goat anti-rat IgG2b (10 µg/ml, 30 minutes, 37°C). Cap formation was stoppedby 2 washes in ice-cold PBS.

Cell spreading was induced by layering IP12-7 cells (1×105 ml inRPMI 1640, 10% fetal calf serum) on Labtek chamber slides, whichhad been precoated with 10 µg/ml IM7. Poly-L-lysine coated chamberslides served as control. Cells were incubated at 37°C for varioustimes. Where indicated cells had been pretreated for 20 minutes at37°C with the following agents: 50 mM 2-deoxyglucose plus 0.04%sodium azid (DOG/azid), 1 µg/ml cytochalasin B, 1 µM nocodazole,80 µM pp2, 100 nM wortmannin, 50 µM Ly294002, 0.5 µM ocadaicacid, 12 mM methyl-β-cyclodextrin or DMSO (carrier).

Flow cytometry and immunofluorescence microscopyFlow cytometry followed routine procedures using 3-5×105 IP12-7cells per sample. Samples were analyzed by a FACSCalibur (BectonDickinson, Heidelberg, Germany).

After cap formation, cells were transferred onto adhesion slides andwere incubated for 15 minutes in PBS, the slides were rinsed oncewith PBS and cells were fixed for 30 minutes in 4% paraformaldehyde(w/v in PBS).

After spreading, slides were gently washed, cells were fixed in4% paraformaldehyde (w/v in PBS) and were permeabilized byincubation for 4 minutes in 0.1% (v/v) Triton X-100. After washingand blocking non-specific binding sites, cells were incubated withthe primary antibody at pretested concentrations (5-10 µg/ml) inPBS/BSA for 60 minutes. Slides were rinsed and subsequentlyincubated for 60 minutes with a fluorochrome-conjugated secondaryantibody. F-actin was stained by an additional 60 minutes incubationwith phalloidin-TRITC (1 µg/ml). After washing 3 times in PBS andonce in H2O, slides were mounted in Elvanol. Digitized images weregenerated using a confocal laser scanning microscope (TS NT, Leica,Germany). For the evaluation of two-color experiments digital imageswere overlaid electronically.

Purification of GEM fractions and subcellular fractionationIP12-7 cells (5×106) were seeded into Petri dishes precoated with eitheranti-CD44 (IM7) or control IgG and incubated for 15 minutes at 37°C.Stimulation was terminated by transferring the dishes to ice andimmediate lysis of the cells. IP12-7 cells (5×106) were lysed for 30minutes in 1 ml of ice-cold TNE-buffer (20 mM Tris-HCl, pH 7.4, 150ml NaCl, 2 mM EDTA, 10 mM NaF, 1 mM Na3VO4) containing 0.5%Triton X-100 and a protease inhibitor cocktail (Boehringer Mannheim).The lysate was adjusted to 40% (w/v) sucrose by mixing with 1 ml 80%(w/v) sucrose made with TNE-buffer. After transfer of the lysate to thecentrifuge tube, 2 ml 30% (w/v) and 1 ml 5% (w/v) sucrose in TNEwas overlaid. Samples were centrifuged for 16-18 hours at 200,000 gat 4°C. Gradient fractions (0.4 ml) were collected from the top.Fractions were analyzed by SDS-PAGE and western blotting. GEM-associated fyn was solubilized in 1% Triton X-100 plus 0.2% saponinin TNE-buffer at 4°C before immunoprecipitation by anti-fyn mAb.

ImmunoprecipitationIP12-cells (5×106) were lysed in ice-cold TNE-buffer (20 mM Tris-HCl, pH 7.4, 150 ml NaCl, 2 mM EDTA, 10 mM NaF, 1 mM Na3VO4)containing either 1% (v/v) Triton X-100 or 1% (v/v) Triton X-100

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1171CD44-mediated cytoskeleton rearrangement

plus 0.2% (w/v) saponin. All lysis buffers contained a proteaseinhibitor cocktail (Boehringer Mannheim). After clarification bycentrifugation for 10 minutes at 10,000 g cell lysates were subjectedto immunoprecipitation.

Lysates were precleared by the addition of 5 µg control antibodyfor 60 minutes followed by incubation with 1/10 volume Protein A-Sepharose for 2 hours at 4°C. Precleared lysates were incubated for60 minutes at 4°C with 2 µg of anti-fyn or control IgG. Protein A-Sepharose was added for an additional 60 minutes. Immunecomplexes were washed 4 times with lysis buffer. Immunoprecipitatedproteins were analyzed by SDS-PAGE, followed by western blotting.

Western blottingLysates were resolved on 7.5% SDS-PAGE under reducing or non-reducing conditions and the proteins transferred to Immobilon Pat 90 V for 1 hour. After blocking the membranes with 3% BSA,immunoblotting was performed by using the indicated antibodies,followed by donkey anti-mouse HRP or donkey anti-rabbit HRP. Blotswere developed with the enhanced chemiluminescence detectionsystem. When the same blot was revealed with different probes,antibody stripping was performed according to the manufacturer’srecommendations.

RESULTS

Cross-linking of CD44 induces cap formation andcell spreadingWe recently have described that CD44 functions as acostimulatory molecule likely by the recruitment of lck and fyn

into the vicinity of the T cell receptor (Föger et al., 2000).Furthermore, it is known that F-actin accumulates at theinterface between the T cell and an antigen presenting cell,which stabilizes this interaction and provides a scaffold forsignaling components required for T cell activation (Penningerand Crabtree, 1999). Since CD44 has been repeatedly reportedto associate with elements of the cytoskeleton, it couldwell have been that engagement of CD44 supports thisrearrangement of the cytoskeleton. To test the hypothesis wefirst evaluated whether CD44 is involved in cap formation.When IP12-7 cells, a TH line which expresses CD44 at a highlevel (Rajnavolgyi et al., 1994), were incubated with anti-CD44 (IM7) and a secondary FITC-labeled antibody for cross-linking, we observed capping of CD44. Cap formation wasassociated with a strong and polarized accumulation of F-actin(Fig. 1A). Furthermore, Rac1, a member of the Rho family ofsmall GTPases, clearly colocalized with CD44 (Fig. 1B). Capformation was also induced by KM81, a CD44-specificantibody blocking the hyaluronan binding site. Although Rac1was enriched in the KM81-induced cap, there remained aconsiderable proportion of free Rac1, i.e. KM81 may not be anactivating antibody (data not shown). Neither Rho nor Cdc42accumulated at the site of the caps.

Cross-linking of CD44, in addition, initiated adhesion,flattening and spreading of IP12-7 cells. This has beenobserved when seeding the cells on IM7-coated plates (Fig.2A). It has not been observed when seeding the cells on KM81-coated plates, KM81 recognizing a distinct CD44 epitope in

Fig. 1.Cap formation induced by CD44 cross-linking. (A) Cap formation and accumulation of F-actin: CD44 receptor caps were induced byincubating IP12-7 cells with soluble anti-CD44 and cross-linking with an FITC-conjugated secondary antibody (top). After fixation andpermeabilization F-actin was visualized by staining with phalloidin-TRITC (middle). A digital overlay is shown at the bottom.(B) Colocalization of the small GTPase Rac1 with CD44 receptor caps: CD44 caps were induced as described (green fluorescence, left panel).Localization of Rac1, RhoA and Cdc42 was evaluated by counterstaining with specific primary antibodies and Texas-red conjugated secondaryantibodies (right panel). Only Rac1 colocalizes with the CD44 cap (digital overlays in the middle panel).

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the HA binding domain. On IM7-coated plates the cellsadhered within minutes and spread over a period of 1 hour asevidenced by the formation of F-actin bundles. Thephenomenon has not been restricted to IP12-7 cells, whichexpress very high levels of CD44, but was also observed withanother T cell line (D10-G4.1) as well as with T cell blasts(Fig. 2B). Spreading depended on a functional cytoskeleton,because cells did neither spread in the presence of the actindisruption agent cytochalasin B nor in the presence ofnocodazole, which disrupts the microtubule system (Fig. 2A).The process was energy dependent. It did not take place at 4°Cnor after pretreatment of the cells with 2-deoxyglucose-azide(Fig. 2A). Finally, as described for cap formation, spreadingwas accompanied by formation of punctuated spots of Rac1,

while Rho and Cdc42 remained distributed throughout thecytoplasm (Fig. 2C).

Signaling elements involved in CD44-inducedcytoskeleton rearrangementPI3 kinase and kinases of the src family have been implicatedin cell adhesion and spreading (Han et al., 1998). In fact, whenIP12-7 cells were pretreated with the src kinase inhibitor pp2,CD44-mediated spreading was inhibited in a dose dependentmanner (Fig. 3A). On the other hand, there was no evidencefor an involvement of PI3 kinase, i.e. spreading appearedunaltered when cells had been preincubated with LY294002 orwortmannin, two inhibitors of PI3-kinase (Fig. 3B). Spreadingwas also independent of serine phosphatase type 1 and 2 as

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Fig. 2.CD44-induced cell spreading. (A) CD44 mediated spreading: IP12-7 cells were seeded on plates coated with BSA, KM81 (anti-CD44recognizing an epitope in the HA binding domain), IM7 (anti-CD44 recognizing an epitope outside the HA binding domain). Where indicatedIP12-7 cells were incubated at 4°C or were pretreated with 2-deoxyglucose-0.04% sodium azide (DOG/azide, energy depleting), cytochalasin B(disruption of actin), nodazole (disruption of microtubules). The light microscope appearance after 1 hour of incubation is shown. (B) Timecourse of cytoskeletal rearrangement during CD44-induced spreading: IP12-7 cells were seeded on IM7-coated plates. Cells were fixed andpermeabilized after the indicated time points and F-actin was stained with phalloidin-TRITC. Cytoskeletal rearrangement by CD44-inducedspreading is shown, in addition, for D10.G4.1 cells (10 minutes) and T cells, which had been cultured overnight in IL-2 (10 U/ml) containingmedium (60 minutes). At the indicated times, no spreading of D10.G4.1 and of T cells has been observed on BSA coated plates (data notshown). (C) Distribution of Rho-family GTPases during CD44-mediated spreading: IP12-7 cells were seeded on IM7-coated plates and werefixed and permeabilized after 60 minutes. The subcellular localization of RhoA, Cdc42 and Rac1 was revealed by indirect immunofluorescencestaining as described above. In the lower right quadrant, cells were double stained with KM81/anti-rat IgG2a-FITC and anti-Rac1/anti-rabbitCy3 for demonstrating directly co-distribution of CD44 and Rac1.

1173CD44-mediated cytoskeleton rearrangement

evidenced by spreading after pretreatment with the inhibitorocadaic acid.

Because CD44-mediated cell spreading was accompaniedby targeting Rac1 to cell protrusions, we next explored whetherRac1 was essential for the CD44-induced rearrangement of thecytoskeleton. IP12-7 cells were transiently transfected withmyc-tagged dominant negative mutant forms of Rac1 (myc-RacN17) and Rho (myc-RhoN19). Expression of the mutantproteins was confirmed by immunoblotting with anti-myc (datanot shown). When myc-Rac1N17 transfected cells, which hadbeen cultured for 1 hour on anti-CD44-coated dishes werestained with phalloidin-TRITC (and counterstained withthe anti-myc antibody) no formation of F-actin fiberscould be seen (Fig. 4). In addition, cells remained roundand did not spread at all. Overexpression of myc-RhoN19did not interfere with spreading and the formation ofactin bundles was, if at all, only slightly reduced. Thus,Rac1 is required for the CD44-mediated cytoskeletalrearrangement, which leads to cell spreading.

CD44-mediated cell spreading is associatedwith CD44 relocalization into lipid raftsSince CD44 functions as a costimulatory molecule likelyby the recruitment of lck and fyn into the vicinity of theT cell receptor (Föger et al., 2000), it became tempting tospeculate that the CD44-induced rearrangement of thecytoskeleton may be associated with a redistribution of

CD44 into lipid-rich rafts. A first hint was obtained by seedingIP12-7 cells on IM7-coated plates after treatment with methyl-β-cyclodextrin, which leads to redistribution of raftscomponents by extracting cholesterol (Xavier et al., 1998). Infact, methyl-β-cyclodextrin-treated IP12-7 cells did not spreadat all neither on substrate (fibronectin) nor on IM7-coatedplates (Fig. 5A). Furthermore, when Triton X-100 lysates ofIP12-7 cells, derived from suspension cultures, wereimmunoprecipitated with fyn, only a small amount ofcoprecipitated CD44 could be detected (Fig. 5B). Instead,when rafts were solubilized by the addition of 0.2% saponin,

Fig. 3. Signaling molecules involved inCD44-induced cell spreading. (A) Thesrc-kinase inhibitor pp2 reduces CD44-mediated spreading in a dosedependent manner: IP12-7 cells werepretreated with various concentrationsof pp2 and seeded on plates coatedwith IM7. The percentage of spreadcells was scored after 1 hour using aphase contrast microscope. (B) CD44-mediated spreading is independent ofPI3 kinase and serine phosphatase type1 and 2: IP12-7 cells were pretreatedwith wortmannin or LY294002 (PI3-kinase inhibitors) or with the DMSOcarrier (negative control) or with ocadaic acid (serine phosphatase type 1 and 2 inhibitor) or with the src kinase inhibitor pp2 (positive control).The light microscope appearance after 1 hour of incubation is shown. Spreading was only prevented by pp2.

Fig. 4. Colocalization of Rac1 is required for CD44-mediatedspreading and F-actin bundle formation. IP12-7 cells weretransiently transfected with plasmids encoding myc-taggeddominant negative mutant forms of Rac1 (myc-RacN17) orRhoA (myc-RhoN19) and were seeded 16 hours aftertransfection on IM7 coated plates. After 1 hour cells were fixedand permeabilized. Transfected cells were detected byimmunofluorescence staining with anti-myc and a Cy2-conjugated secondary antibody (green, left panel). F-actin wasvisualized by staining with phalloidin-TRITC (red, rightpanel). The digital overlays are shown in the middle panel.Rac1 negative mutant transfected cells do not spread and donot form F-actin bundles.

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significantly more p59fyn could be immunoprecipitated and theamount of coprecipitated CD44 was strongly increased.Control precipitates did neither contain p59fyn nor CD44. Thedata suggest that in T cells the fraction of CD44 whichconstitutively associates with p59fyn is mainly located in therafts.

To control whether cross-linking of CD44 is associated witha redistribution of CD44 and CD44-associated p59fyn into lipidrafts, IP12-7 cells were stimulated on plates coated with anti-CD44 or control IgG. After solubilization and density sucrosegradient ultracentrifugation to separate the low density GEMfractions, it was in particular a tyrosine phosphorylated proteinwith a molecular mass of approximately 60 kDa, which wasaugmented in the raft fractions after stimulation by anti-CD44(Fig. 6A). Stripping the blot and reprobing with anti-fynrevealed the same pattern (Fig. 6B), i.e. quantification bydensitometry revealed a ratio of 2.3:1 pixel in lysates of anti-CD44 stimulated cells as compared to control lysates in theGEM fractions. Since pp60 and fyn displayed the samemobility in the polyacrylamide gel, it is very likely that pp60is identical to fyn. This was controlled by immunoprecipitatingthe pooled GEM and Triton X-100 soluble fractions with anti-fyn. After SDS-PAGE of the immunoprecipitates they wereblotted with anti-phosphotyrosine and anti-fyn (Fig. 6C). Infact, pp60 was recovered in the anti-fyn precipitate, i.e. isidentical to p59fyn. Thus, engagement of CD44 leads to aredistribution of fyn into the rafts. However, it should be notedthat targeting of fyn into the lipid rafts was not associated withan increase in phosphorylation, i.e. the increase in tyrosinephosphorylation correlated with the increase in the amount offyn. Besides of fyn, p56lck also was found to be redistributedto the rafts (data not shown). Importantly, CD44, too, wasconsistently found to be enriched in the rafts (ratio ofdensitometry values of GEM fractions of unstimulated cells toGEM fractions of stimulated cells: 1:5.4) (Fig. 6D). To controlfor a direct association of fyn with CD44 in the lipid rafts,

pooled Triton solubilized fractions and GEM fractions wereimmunoprecipitated with anti-CD44 and anti-fyn. After SDS-PAGE the immunoprecipitates were blotted with anti-CD44and anti-fyn. In the Triton soluble fraction only a small amountof fyn coprecipitated with CD44, while in the GEM fractioncomparable amounts of fyn were precipitated by anti-fyn andanti-CD44, i.e. in the GEM fraction the vast majority of fyn,indeed, was found to be associated with CD44 (Fig. 6E).

Taken together, engagement of CD44 initiates F-actinbundle formation accompanied by a redistribution of CD44 andthe associated tyrosine kinases into the rafts, i.e. into theneighborhood of the peptide-MHC engaged TCR.

DISCUSSION

Efficient activation of T cells requires the engagement ofcostimulatory molecules (Chambers and Allison, 1997; Croftand Dubey, 1997). Recently, it has been debated whether thesecostimulatory molecules exert their function by stabilizing theTCR-MHC interaction and/or by recruiting signal transductionmolecules towards the TCR/CD3 complex (Shaw and Dustin,1997; Wülfing and Davis, 1998; Viola et al., 1999). Ourprevious analysis of a costimulatory function of CD44 in T cellactivation supported the latter hypothesis, i.e. costimulation bythe recruitment of the src-family kinases p59fyn and p56lck

(Föger et al., 2000). This finding raised the question as to themechanism by which CD44 strengthened signaling via theTCR/CD3 complex. There were at least 3 possibleexplanations: (i) CD44 functions as an adhesion moleculewhich allows for a prolonged contact between the TCR and itsligand; (ii) cross-linking of CD44 initiates reorganization of thecytoskeleton towards a focal actin-scaffold, which is requiredfor effective T cell activation (Wülfing and Davis, 1998;Howard and Watts, 1994; Dustin et al., 1998; Valitutti et al.,1995); (iii) cross-linking of CD44 leads to its redistribution in

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Fig. 5. Involvement of rafts in CD44 mediatedspreading. (A) Spreading induced by CD44cross-linking requires intact rafts: Untreated andmethyl-β-cyclodextrin (βCD) pretreated IP12-7cells were seeded on BSA- or IM7-coated plates.To control for the efficacy of cholesterolsequestration, IP12-7 were also seeded onfibronectin (Fn). IP12-7 cells do not spread onfibronectin coated plates after methyl-β-cyclodextrin treatment. The light microscopeappearance after 1 hour of incubation is shown.(B) Coimmunoprecipitation of CD44 withp59fyn: IP12-7 cells in suspension culture wereharvested and were detergent solubilized by 1%Triton X-100 (lanes 1 and 2) or 1% Triton X-100plus 0.2% saponin (lanes 3 and 4). Lysates wereimmunoprecipitated with control IgG (lane 1 and3) or anti-fyn (lane 2 and 4). Precipitates wereresolved by SDS page and immunoblotted withanti-CD44 (IM7) (upper blot). After stripping theblot was reprobed with anti-fyn (lower blot). Thepositions of CD44 and p59fyn are indicated by arrowheads. Detection of Ig due to binding of the secondary HRP-conjugated antibody isindicated by a closing square bracket. Molecular mass markers are shown in kDa.

1175CD44-mediated cytoskeleton rearrangement

the membrane in such a way that it neighbors the TCR/CD3complex, which would facilitate an efficient delivery of theconstitutively CD44-associated PTKs fyn and lck. In the lattercase it became of special interest, whether CD44 would be

driven towards glycolipid-enriched microdomains, which alsogather the TCR during the activation process (Wülfing andDavis, 1998; Xavier et al., 1998; Viola et al., 1999; Kosugi etal., 1999; Montixi et al., 1998). We now report on a CD44-

Fig. 6. Cross-linking of CD44 induces the redistribution of p59fyn intoglycolipid-enriched microdomains. IP12 cells were stimulated for 15 minutesat 37°C on IM7 or control IgG coated plates. Cells were lysed and lysates weresubjected to sucrose gradient ultracentrifugation collecting the 12 topfractions. (A and B) Enrichment of a tyrosine phosphorylated protein in theGEM fractions: Fractions were resolved by SDS-PAGE (reducing conditions)and were blotted with anti-phosphotyrosine (A) and after stripping with anti-fyn (B). GEM fractions 1-5 of lysates of IP12-7 cells seeded on IM7-coatedplates contain an increased amount of a 60 kDa tyrosine phosphorylatedprotein which comigrates with p59fyn. (C) Verification of pp60 as p59fyn: GEMfractions (2-4) and Triton X-100 soluble fractions (10-12) were pooled andimmunoprecipitated with anti-fyn. The immunoprecipitates were resolved bySDS-PAGE (reducing conditions) and were blotted with anti-phosphotyrosine(upper row) and after stripping with anti-fyn (lower row). Pp60/p59fyn aremarked by an arrow head, Ig is indicated by a closing square bracket. While inthe Triton-soluble fractions comparable amounts of pp60/p59fyn wereimmunoprecipitated from unstimulated and CD44 stimulated IP12-7 cells,pp60/p59fyn was significantly enriched in the GEM fraction of CD44-stimulated IP12-7 cells. (D) Redistribution of CD44 in the GEM fraction:Fractions 1-12 after sucrose gradient centrifugation were dissolved by 7.5%

SDS-PAGE under non-reducing conditions and immunoblotted with IM7. Upper row: control IgG stimulated IP12-7 cells; lower row: IM7stimulated IP12-7 cells. Molecular mass markers are shown in kDa. Only after stimulation with IM7 a significant amount of CD44 wasrecovered in the GEM fractions. (E) Verification of the CD44-fyn association in GEM: GEM fractions and Triton X-100 soluble fractions werepooled and immunoprecipitated with anti-CD44 or anti-fyn. The immunoprecipitates were resolved by SDS-PAGE and were blotted with anti-fyn (upper row). In the GEM fraction equal amounts of fyn were recovered after precipitation with anti-fyn and anti-CD44. Hence, in the GEMfraction the vast majority of p59fyn was associated with CD44. The immunoprecipitates with anti-CD44 were stripped and blotted with anti-CD44 (lower row). As shown above, CD44 was enriched in the GEM fraction.

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mediated rearrangement of the actin cytoskeleton and aredistribution of CD44 as well as of the associated kinasesp59fyn and p56lck into lipid rafts.

The TCR has a relatively low affinity for its peptide-MHCligand (Davis et al., 1998). Furthermore, T cells and APCs havea tendency to repel each other owing to their net negativesurface charge (Springer, 1994). On the other hand, T cellactivation requires a sustained contact (Davis et al., 1998). Theprime candidates to provide the required attractive forces areadhesion molecules (Dustin and Springer, 1989; Croft andDubey, 1997; Valitutti et al., 1995). Thus, CD44, a wellcharacterized adhesion molecule, could well function as acostimulatory molecule by strengthening the contact betweenT cell and APC either directly or by potentiating integrinactivity as observed after triggering of CD44 on T cells(Koopman et al., 1990). However, CD44 capping, spreadingand, as already reported, costimulation (Föger et al., 2000)depended on the epitope recognized by the cross-linkingantibody. These findings argue against ligand binding as theexclusive function of CD44.

Recently it has been demonstrated that TCR clusteringrequires actin polymerization in the T cell (Wülfing and Davis,1998; Penninger and Crabtree, 1999; Dustin et al., 1998;Valitutti et al., 1995). It has been suggested that in the firstinstance signals have to be delivered which initiate actinpolymerization and accumulation of cortical actin at thecontact area, which could serve as a scaffold for thereorganization of membrane microdomains as well as for theformation of the multimolecular signaling complexes requiredfor T cell activation (Wülfing and Davis, 1998; Penninger andCrabtree, 1999). The importance of actin reorganization forreceptor clustering and T cell activation has been demonstratedby the treatment of T cells with cytochalasins, which disruptactin filaments: TCR capping is inhibited, T cells do not changeshape and activation is significantly impaired (Holsinger et al.,1998; Kong et al., 1998; Valitutti et al., 1995). Cross-linkingof CD44 via IM7 (but not via KM81) induced profoundmorphological changes, like increased adhesion, flattening andspreading accompanied by the formation of actin bundles.Accordingly, capping of CD44 by IM7 was accompanied byaccumulation of F-actin at the site of the cap. Taking thesefeatures, it becomes very likely that CD44 is a potent candidatefor mediating the cytoskeletal rearrangements required for theinitiation of T cell activation. Our finding also provided thebasis for a detailed analysis of the involved signaling elements.Although this analysis has yet to be completed, a variety ofcandidate molecules could be identified or excluded by theuse of specific inhibitors. Thus, we could demonstrate theinvolvement of src-family kinases, which also have beendescribed to be involved in integrin mediated spreading andmigration (Price et al., 1998). Since CD44 is constitutivelyassociated with fyn and lck, it is likely that these kinases maybe involved in the very proximal signaling leading tocytoskeletal restructuring. PI-3 kinase has been reported toinfluence actin dynamics following engagement of CD2 andCD28 (Shimizu et al., 1995). However, it apparently is notinvolved in CD44 mediated actin reorganization. The Rhosubfamily of small GTPases are considered as key regulatorsof the actin cytoskeleton (Mackay and Hall, 1998; Hall, 1998).By immunofluorescence as well as by the use of dominantnegative mutants we could clearly demonstrate the involvement

of Rac1 in CD44-induced actin polymerization, which recentlyhas also been described for hyaluronic acid binding of CD44(Oliferenko et al., 2000). Besides of its well characterized rolein mediating actin cytoskeletal changes in fibroblasts (Mackayand Hall, 1998; Hall, 1998), Rac has also been implicated inintegrin-mediated adhesion and spreading of T lymphocytes(D’Souza et al., 1998). Two downstream targets of Rac whichlikely play a central role in actin polymerization arephophatidyl-4-phosphate kinase and p65PAK (Hartwig et al.,1995). The product of phophatidyl-4-phosphate kinase,phophatidyl-4,5-biphosphate is known to affect actin filamentassembly (Hartwig et al., 1995), p65PAK is supposed tointeract with a molecular complex that controls actinpolymerization (Price et al., 1998). Whether p65PAK isinvolved directly in the activation of the JNK pathway is stilla matter of debate (Brown et al., 1996; Tapon et al., 1998).With respect to cross-linking of CD44 which induced Rac1activation, we did not observe c-jun phosphorylation. Thisargues against CD44 affecting T cell activation directly via aRac1 – p65PAK – JNK signaling pathway. We also found noevidence for an involvement of ERM proteins, which have beendescribed to anchor actin to cell surface receptors as aprerequisite for Rho and Rac to induce cytoskeletal changes(Mackay et al., 1997). We could, however, neither detect ezrinnor RhoGDI in CD44 immunoprecipitates of T cells (data notshown). Finally, it should be mentioned that the CD44-inducedmorphological changes also involved the microtubular system.This is not surprising, since in many systems the cortical actinand microtubules act in concert. It has been described thatmicrotubule growth, activation of Rac1 and actinpolymerization functionally cooperate (Waterman-Storer et al.,1999), an observation which is fully in line with our findings.

Although we have discussed the CD44-induced cytoskeletalreorganization particularly in view of T cell activation, itshould be mentioned that T cell adhesion, flattening andspreading may also be important for homing, rolling,extravasation and migration through the space of theextracellular matrix. CD44 is known to be involved in all ofthese processes (Borland et al., 1998).

By reorganization of the cytoskeleton, which would allowfor an increase in the contact area between the T cell and theAPC, CD44 could strengthen signaling via the TCR/CD3complex. There remains the question whether thereorganization of the cytoskeleton is accompanied by therecruitment of receptors and/or signaling molecules to the zoneof contact, as has been described for the TCR (Shaw andDustin, 1997; Wülfing and Davis, 1998; Xavier et al., 1998;Kosugi et al., 1999; Montixi et al., 1998; Grakoui et al., 1999;Moran and Miceli, 1999). Several lines of evidences stronglysupport the hypothesis. It has been described for the humansystem (Dianzani et al., 1999) and we have described for themurine system, too, that CD44 is constitutively associated withlck and fyn, which phosphorylate multiple ITAM motifs of theTCR/CD3 complex at the initiation of activation. We also couldidentify a binding motif of CD44 which is responsible for theassociation with src-kinases (Rozsnyay, 1999). An efficientphosphorylation of both the TCR/CD3 complex and ZAP70 bylck and fyn has also been described for the costimulatorymolecules CD4, CD2 and CD28 (Davis and van der Merwe,1996; Holdorf et al., 1999). Furthermore, lck and fyn have alsobe shown to be essential as adaptor proteins, important for the

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1177CD44-mediated cytoskeleton rearrangement

assembly of a transduction-competent signaling complex atlatter stages of T cell activation. It also has been suggested thatcellular stimulation via CD44 may proceed through thesignaling machinery of GEMs, because CD44 has been foundto selectively associate with active src-family protein tyrosinekinases in these microdomains (Ilangumaran et al., 1998). Thehypothesis is fully in line with our observations that CD44-induced spreading is prohibited by sequestration of cholesterol,that CD44 associated fyn is particularly enriched in GEMs andthat cross-linking of CD44 is associated with a redistributionof CD44, fyn and lck into lipid rafts. Accordingly, it has beendescribed that costimulation by CD28 or LFA-1 initiates anactin-myosin driven directional transport of protein and lipiddomains to the T cell – APC contact zone (Wülfing and Davis,1998), coengagement of CD28 and TCR/CD3 complexrecruiting all GEMs to the contact area (Viola et al., 1999).

Taken together, cross-linking of CD44 induces areorganization of the actin cytoskeleton, which involves themicrotubule system and leads to T cell adhesion, flatteningand spreading. The reorganization of the cytoskeleton isaccompanied by a redistribution of CD44 and the associatedPTKs fyn and lck into lipid rafts. Because of these features andthe findings that i. engagement of the TCR also leads to itsredistribution in lipid rafts, (ii) CD44 functions as acostimulatory molecule by lowering the threshold for signaltransduction via the TCR/CD3 complex, we interpret our datain the sense that it is the CD44-induced reorganization of thecytoskeleton and the associated redistribution into GEMs whichprovide the basis for the costimulatory function of CD44.

This work was supported by the DeutscheForschungsgemeinschaft, grant no Zo40/5-3 (MZ). We cordially thankDr E. Rajnavolgyi. L. Eotvos University, God, Hungary, for thegenerous supply of the T cell line IP12-7 and Prof. P. Boquet,INSERM U452, Nice, France, for kindly providing us with expressionvectors encoding myc epitope tagged dominant negative mutant formsof RhoA and Rac1.

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