2175Journal of Cell Science 108, 2175-2186 (1995)Printed in Great Britain © The Company of Biologists Limited 1995

Immunomorphological characterization and effects of 12-(S)-HETE on a

dynamic intracellular pool of the αIIbβ3-integrin in melanoma cells

J. Tímár1, R. Bazaz2, V. Kimler3, M. Haddad3, D. G. Tang2, D. Robertson4, J. Tovari1, J. D. Taylor3 and K. V. Honn2,5,*11st Institute of Pathology and Experimental Cancer Research, The Semmelweis Medical University, Budapest, HungaryDepartments of 2Radiation Oncology, Chemistry and Pathology, and 3Biological Sciences, Wayne State University, Detroit MI 48202, USA4Institute of Cancer Research, Royal Cancer Hospital, Haddow Labs, EM Unit, Belmont, Sutton, Surrey, UK5Division of Cancer Biology, Gershenson Radiation Oncology Center, Harper Hospital, Detroit, MI 48201, USA

*Author for correspondence at: Department of Radiation Oncology, Cancer Biology Division, 431 Chemistry, Detroit, MI 48202, USA

In metastatic B16a murine melanoma cells, αIIbβ3 integrinwas shown to be one of the key adhesion molecules respon-sible for matrix adhesion and spreading. Upon stimulation,αIIbβ3 can be upregulated at the cell surface due to translo-cation of the receptor to the plasma membrane from anintracellular pool. Here we have characterized this integrinpool as a tubulovesicular structure (TVS) corresponding toendosomes. TVS was found to be associated temporarilywith microtubules and intermediate filaments especiallyafter protein kinase C (PKC) stimulation with a lipoxy-genase metabolite of arachidonic acid, 12-(S)-hydroxyeico-satetraenoic acid [12-(S)-HETE]. After PKC stimulation,the predominantly vesicular TVS became elongated andαIIbβ3 appeared at the apical plasma membrane andmicrovilli. Disruption of either the microtubules or inter-

mediate filaments prevented the 12-(S)-HETE effect bothon vesicular to tubular transition of TVS as well as onsurface expression of this integrin. The connection with theGolgi system of the integrin-containing TVS was proved bya Golgi-inhibitor (brefeldin A) pretreatment, whichprevented the PKC-stimulation-induced TVS elongationand subsequent receptor-upregulation at the cell surface.After a soluble ligand binding (mAb to the αIIbβ3 complex)the surface receptor endocytosed back to the TVS indicat-ing the presence of a dynamic, cytoskeleton associatedintegrin pool in melanoma cells.

Key words: 12(S)-HETE, αIIbβ3, tubulovesicular structure,melanoma

SUMMARY

INTRODUCTION

Integrins are the principal matrix receptors which provide anessential link between the extracellular milieu and thecytoskeleton (Albeda and Buck, 1990). Their association withcytoskeletal elements at the plasma membrane is well known(Burridge, 1986); however, little is known about their associ-ation with intracellular organelles once synthesized in theendoplasmic reticulum (ER), glycosylated in the Golgi systemand translocated to the cell membrane on cytoskeletal proteintracks. Leukocytes have an intracellular compartment calledadhesomes, which contain receptors involved in cellularadhesions (Singer et al., 1989). Adhesomes, as a result of theirassociation with actin (Singer et al., 1989), translocate to theplasma membrane, where the adhesion receptors are immobi-lized by cytoskeletal proteins; i.e. α-actinin, talin and vinculin.These proteins link the adhesomes to actin filaments to formfocal adhesions or intercellular junctional complexes (Luna,1991).

Unoccupied receptors or soluble ligand-bound receptors atthe plasma membrane are continously endocytosed into anintracellular pool and later degraded by lysosomes (Hopkins,

1986). The presence of a cycling pool for α5β1 and αIIbβ3 hasbeen demonstrated in fibroblasts (Molnar et al., 1987), CHOcells (Raub and Kuentzel, 1989) and platelets (Wenzel-Drake,1990) respectively. However, the nature of this pool and itsrelation to the cytoskeleton is not yet known.

The integrin αIIbβ3, the principal matrix receptor of platelets,is a predominant matrix receptor in some rodent and humantumor cells (Chang et al., 1991, 1992; Chen et al., 1992;McGregor et al., 1989). In these cells, αIIbβ3 was localized tofocal adhesion sites (Chen et al., 1992; Chopra et al., 1992)and was found to be associated with vinculin (Tímár et al.,1992). In B16a melanoma cells, we found an intracellular poolof αIIbβ3 from which integrin molecules translocate to the cellsurface (Chopra et al., 1991). We also demonstrated in murinetumor cells that the intracellular translocation of this integrinto the plasma membrane and its upregulation to the cell surfaceis modulated by a 12-lipoxygenase (LOX) metabolite ofarachidonic acid, i.e. 12-(S)-HETE; Chopra et al., 1991). The12-(S)-HETE action on integrin upregulation appears to bedependent upon both microfilament and intermediate filamentintegrity (Chopra et al., 1991).

In this study we have characterized the intracellular pool of

2176 J. Tímár and others

αIIbβ3 integrin in B16a melanoma cells using immunomor-phological techniques. We found that the majority of the intra-cellular integrin is localized to a tubulovesicular compartment(TVS) most probably corresponding to endosomes. Integrin-containing TVS appears in two forms. In the unstimulated stateit is a predominantly vesicular compartment while upon stim-ulation with 12-(S)-HETE (a physiological activator of proteinkinase C; Liu et al., 1991) it is transformed to tubular struc-tures which become associated with microtubules and inter-mediate filaments. After soluble ligand binding, surface αIIbβ3is endocytosed into the same cytoplasmic TVS compartmentindicating the existence of a dynamic intracellular pool ofαIIbβ3 in melanoma cells.

MATERIALS AND METHODS

AntibodiesMonoclonal antibody AP-2 (IgG) raised in mice against humanplatelet αIIbβ3 was a generous gift from Dr T. J. Kunicki (ScrippsResearch Institute, LaJolla CA), while mAb 10E5 (IgG2) was kindlyprovided by Dr B. S. Coller (Stony Brook, NY) (Pidard et al., 1983).The specificity and crossreactivity of these antibodies for murineαIIbβ3 has been described (Chen et al., 1992). Antiserum to mousevimentin produced in goat was obtained from ICN Immunologicals(Lisle, IL). Biotinylated goat anti-mouse IgG and anti-rabbit IgG wasfrom Boehringer (Indianapolis, IN), rabbit anti-goat IgG-TRIC wasfrom Sigma (St Louis, MO) and Streptavidin-FITC/Texas Red, goatanti-mouse IgG conjugated to 5 nm gold and Streptavidin conjugatedto 15 nm gold were from Amersham (Arlington Heights, IL).

PKC stimulation12-(S)-HETE (Cayman Chemical Co., Ann Arbor, MI) was dilutedfrom 0.3 mM stock solution in EtOH and was used at a final concen-tration of 0.1 µM in serum-free MEM to stimulate PKC in adherentor suspended tumor cells as previously described (Liu et al., 1991).Calphostin C (UBI, Lake Placid, NY) was used as a selective PKCinhibitor (Kobayashi et al., 1989) to pretreat the cells before stimula-tion with 12-(S)-HETE. The inhibitor was dissolved in serum-freeMEM and was applied to the cells exposed to light at 37°C for 10minutes.

Brefeldin A treatmentAdherent or suspended tumor cells were treated with brefeldin A(Gibco/BRL) at concentrations of 5-10 µg/ml for 60-120 minutes at37°C before immunocytochemistry.

Cytoskeleton disruptionAdherent tumor cells were pretreated with (Eckert, 1986) 3 µg/mlnocodazole (Sigma) for 1 hour or with 10 µM acrylamide (ICN,Cleveland, OH) for 4 hours as previously described (Eckert, 1986;Tímár et al., 1993).

Immunofluorescent microscopyDetection of integrin αIIbβ3 at the cell surfaceMonolayers were fixed with 2% paraformaldehyde/HBSS for 15minutes, washed in HBSS, incubated with non-immune goat serumdiluted 1:2 in PBS for 20 minutes and then incubated with mAbAP2or MOPC21 at the same concentration (50 µg/ml; diluted in HBSS/3%BSA) for 60 minutes at 37°C. After washing in HBSS (3×), the boundIgG was detected by biotinylated goat anti-mouse IgG (Boehringer)at a dilution of 1:20 in HBSS/3% BSA for another hour. The wholecomplex was visualized by incubating the cells with FITC-Strepta-vidin conjugate (Amersham) at 10 µg/ml dilution in HBSS/3% BSAfor 1 hour.

Detection of intracellular integrin αIIbβ3

Monolayers were fixed and permeabilized by MetOH/acetone as inthe case of vimentin labeling, washed in HBSS, incubated with non-immune goat serum diluted 1:2 in PBS for 20 minutes and thenincubated with mAbAP-2 or MOPC21 at the same concentration (50µg/ml; diluted in HBSS/3% BSA) for 60 minutes at 37°C. Afterwashing in HBSS (3×), the bound IgG was detected by biotinylatedgoat anti-mouse IgG (Boehringer) at a dilution of 1:20 in HBSS/3%BSA for another hour. The whole complex was visualized by incu-bating the cells with FITC-Streptavidin conjugate (Amersham) at 10µg/ml dilution in HBSS/3% BSA for 1 hour.

Internalization of surface integrin αIIbβ3

B16a cells adherent to coverslips, were washed (3× 5 minutes) inserum-free RPMI and incubated with mAbAP-2 at a concentration of20 µg/ml for 15 minutes at 37°C to allow internalization of theantibody-receptor complex (Chopra et al., 1991). Control cells wereincubated with mAb MOPC21 applied at the same concentration. Bothsamples were washed (3× 5 minutes) in HBSS and then fixed and per-meabilized in MetOH as in the case of vimentin labeling. Cells thenwere washed (3× 5 minutes) in HBSS, blocked with non-immune goatserum and the intracellular mAb was detected as in the case of intra-cellular integrin detection using FITC labeled secondary antibody.

Parallel internalization of surface integrin αIIbβ3 and transferrinB16a cells adherent to coverslips were washed in serum-free RPMIand incubated with human transferrin (100 µg/ml) and AP-2 mAb (20µg/ml) at 4°C in RPMI for 30 minutes. After incubation, coverslipswere washed in serum-free RPMI and were incubated at 37°C for 0-15 minutes. The coverslips were washed in HBSS (2×) and theadherent cells were fixed in MetOH at −20°C for 5 minutes and inacetone at −20°C for 5 seconds. After washing in HBSS (2×) the cov-erslips were blocked in normal goat serum (dilution 1:5 in HBSS).The intracellular transferrin was detected by incubating the coverslipswith goat anti-transferrin IgG (1:50 dilution in HBSS) for 60 minutesfollowed by rabbit anti-goat IgG-biotin conjugate (dilution 1:40 inHBSS) and Streptavidin-Texas Red. This was followed by extensivewashings in HBSS and repeated blocking with normal goat serum.The internalized integrin-AP-2 complex was detected by goat anti-mouse IgG-biotin conjugate and Streptavidin-FITC as above.

MountingCoverslips were washed in HBSS (3×) and then in distilled water (3×)and mounted on glass slides using Citifluor (Ted Pella Inc., Redding,CA) as a quenching medium. All negative controls demonstratedminimal background fluorescence.

Morphometry by fluorescence imaging (ACAS)Labeled adherent tumor cells on coverslips were analyzed for fluo-rescence intensity using an ACAS 470 argon laser-fluorescenceimaging cytometer (Meridian Instruments, Okemos, MI). Theintensity of fluorescence (FITC) was determined at cell-populationlevel (200-500 cells) using automated digital imaging. FITC wasexcited with a laser beam at 488 nm and the emission was measuredabove 530 nm using a 530/30-nm longpass filter. The fluorescenceintensity of each single point was recorded and displayed as a color-coded image. Fluoresence intensity is given in arbitrary units. At leastfour randomly selected areas were analyzed at each condition and theexperiments were run in duplicates.

Flow cytometryB16a cells were incubated in suspension in serum-free MEM at 37°Cfor 30 minutes, and then fixed in 2% paraformaldehyde/HBSS for 15minutes, washed and labeled for surface expression of αIIbβ3 usingmAbAP-2 as in the case of surface labeling. Measurement wasperformed as previously described (Chopra et al., 1991).

2177Effect of 12-(S)-HETE on αIIbβ3 in melanoma cells

Transmission electron microscopic immunocytochemistrySurface labeling for integrin αIIbβ3

B16a cells were allowed to adhere to plastic for 4 hours, fixed in situwith 1% PFA/0.2% glutaraldehyde/HBSS for 30 minutes at roomtemperature, washed for 5 minutes in HBSS (3×) and then incubatedwith non-immune goat serum diluted 1:10 with HBSS for 10 minutesto block Fc receptors. Surface αIIbβ3 was identified using mAb AP2at a concentration of 20 µg/ml in HBSS (1 hour). After washing for5 minutes in HBSS (3×), cells were incubated with GAMG5 (Janssen)diluted 1:10 with HBSS for 1 hour. Finally cells were washed againfor 5 minutes with HBSS (3×) and postfixed in situ with 2% glu-taraldehyde/HBSS for 30 minutes and 1% OsO4/HBSS for 1 hour.The cell monolayers were dehydrated in situ with an ascending EtOHseries, washed with propylene oxide and the detached cell layerembedded into EPON (Ted Pella). Ultrathin sections were obtainedwith an Ultracut microtome using a diamond knife and contrastedusing uranyl acetate and lead citrate. Photographs were taken in aPhilips 301 transmission electron microscope operating at an accel-erating voltage of 80 kV.

Intracellular labeling for integrin αIIbβ3

Prefixed B16a cells (1% paraformaldehyde/0.2% glutaraldehyde/HBSS) were permeabilized with 0.25% Triton X-100 diluted inPHEM-buffer for 30 minutes at room temperature. Cells were washedfor 5 minutes in HBSS (3×) and incubated in non-immune goat serumdiluted 1:10 in HBSS for 15 minutes. Intracellular αIIbβ3 was detectedby incubating the cell layer in mAbAP2 (20 µg/ml) dissolved inHBSS/0.05% Triton/0.1% gelatin overnight at 4°C. After washing for5 minutes in HBSS (3×), cells were incubated in goat anti-mouse IgG-POX conjugate diluted 1:20 with HBSS/0.05% Triton/0.1% gelatinfor 2 hours at room temperature. After washing in HBSS (3× 5minutes) cell layers were incubated in DAB (1 mg/ml) diluted in 0.1M Tris-HCl, pH 7.4, containing 0.03% H2O2 for 30 minutes at roomtemperature. After washing again with HBSS, cells were postfixedwith 2% glutaraldehyde/HBSS/4% tannic acid for 1 hour and 1%OsO4/HBSS for 1 hour. Dehydration and embedding of the cell layerwas performed as described above as well as ultrastructural observa-tion.

Low temperature embedding and immunolabeling of ultrathinsections (Robertson et al., 1992)B16a cells were fixed in situ using 4% paraformaldehyde/HBSS for30 minutes, washed in HBSS (3× 5 minutes) then were processed inCS-Auto (Reichert) apparatus, where dehydration in EtOH took placeat progressively lower temperatures (PLT) (from 4°C down to −50°C). Embedding was done at −50°C using Lowicryl HM-20 resin(infiltration for 12 hours and polymerization under UV light for 48hours). Ultrathin sections were made using an Ultracut microtome anda diamond knife and sections were mounted onto gold grids. Sectionsfirst were incubated in non-immune goat serum (diluted 1:2 in HBSS)for 30 minutes at room temperature. Integrin αIIbβ3 molecules werelabeled with mAbAP2 at a concentration of 20 µg/ml diluted inHBSS/3% BSA for 1 hour. After washing for 5 minutes in HBSS/3%BSA (3×), sections were reincubated with GAMG5 diluted 1:10 withHBSS/3% BSA for 1 hour. Sections were washed in HBSS/3% BSA(3× 10 minutes), then in distilled water (3× 10 minutes) and were con-trasted using lead citrate.

Whole mount transmission electron microscopy (WMTEM)B16a cells were seeded on Formvar- and carbon-coated 200 meshgold grids at a density of 40,000 cells/200 µl complete MEM andcultured overnight before experiments. Cells were fixed in 1%paraformaldehyde/1.25% glutaraldehyde/0.12M Na-cacodylatebuffer, pH 7.4, for 1 hour and washed (2×) in cacodylate buffer. Fixedcells were treated with 1 mg/ml taxol (NCI, Bethesda, MD) and 10mg/ml phalloidin (Sigma) both diluted in cacodylate buffer, for 20

minutes each and fixed again in 3% glutaraldehyde/4% tannicacid/0.12 M Na-cacodylate buffer for 1 hour. Postfixation wasperformed in 1% osmium tetroxide/0.12 M Na-cacodylate buffer(Electron Microscopy Sciences), pH 7.5, for 15 minutes. Cells werewashed (2×) in cacodylate buffer and (3×) in distilled water andstained with 5% (w/v) aqueous uranyl acetate (Baker Chemical Co.,Phillisburg, NJ) for 45 minutes followed by distilled water rinses (3×).Dehydration was performed with a graded series of EtOH followedby 3× 10 minutes Freon TF-113 (Matheson Gas Products, Twinsburg,OH). Cell monolayers on grids were then critical-point dried (ModelSPC-1500, Bomar, Tacoma,WA) and photographed using a Philips201 or 301 transmission electron microscope at an acceleratingvoltage of 80-100 kV.

Immuno whole mount transmission electron microscopy (I-WMTEM) of integrin αIIbβ3

B16a cells were seeded on carbon-coated Formvar-gold grids for 12hours in serum-containing medium and fixed in 1% paraformalde-hyde/0.1% glutaraldehyde/HBSS for 10 minutes at room temperature.After washing in HBSS for 10 minutes (3×) the Fc receptors wereblocked by incubating the cells in non-immune goat serum diluted 1:2in HBSS. Surface αIIbβ3 was detected by incubating the grids in mAbAP2 (20 µg/ml in HBSS) for 1 hour, washed for 10 minutes (3×) inHBSS and reincubated in biotinylated goat anti-mouse IgG diluted1:20 in HBSS. After washing in HBSS buffer, the cells were incubatedin Streptavidin-G15 diluted 1:10 in HBSS/3% BSA for 1 hour. Thegrids were extensively washed in HBSS (3× 5 minutes) and refixedin 2% glutaraldehyde/HBSS for 1 hour, dehydrated in EtOH, criticalpoint-dried and examined as above.

Internalization of surface αIIbβ3

B16a cells adherent to Formvar-coated grids were washed in serum-free RPMI at 4°C and incubated in AP2 antibody containing RPMIat 4°C (20 µg/ml) for 15 minutes. After washings in RPMI (2× 5minutes), they were reincubated with biotinylated goat anti-mouseIgG diluted 1:20 in RPMI for 15 minutes at 4°C. After washing (3×5 minutes) in cold RPMI the cells were reincubated at 4°C with Strep-tavidin-G15 diluted 1:10 in serum-free RPMI for 15 minutes. Thegrids were washed for 5 minutes (3×) at 4°C and incubated at 37°Cto internalize the integrin-antibody-gold complex for 15-30 minutes.After the incubation the grids were fixed and processed as in the casedescribed for routine WMTEM preparations. Control samples wereincubated with mAb MOPC21.

Internalization of peroxidaseB16a cells adherent to Formvar-coated grids were washed (2×) inserum-free RPMI for 5 minutes at room temperature and incubatedwith horseradish peroxidase (HPOX) diluted in RPMI at a concen-tration of 1 mg/ml for 15 minutes at 37°C. After washing in serum-free RPMI for 5 minutes, the grids were fixed in paraformalde-hyde/glutaraldehyde mixture as in the case of surface labeling for 1hour. After washing with HBSS for 5 minutes (3×) the cells wereincubated in 0.1 M Tris-HCl, pH 7.5, containing 1 mg/ml DAB (Poly-science) and 0.03% H2O2 for 30 minutes at room temperature. Thegrids were washed (3× 5 minutes) with HBSS, postfixed in 1%OsO4/HBSS for 1 hour and processed as described for WMTEM.HPOX was omitted from negative control preparations.

RESULTS

B16a cells were cultured on fibronectin-coated coverslips, per-meabilized and labeled with mAbAP-2 for intracellular αIIbβ3and examined by fluorescent microscopy. mAbAP-2 recog-nizes the αIIbβ3 integrin heterodimeric protein complex but notthe individual monomer subunits (Pidard et al., 1983).Therefore intracellular labeling with this mAb reveals

2178 J. Tímár and others

Fig. 1. Immunofluorescent localization of intracellular αIIbβ3 in non-stimulated B16 cells spread on fibronectin. Note the clear vesicularlabeling around the nucleus (arrowhead) and rare signal at the cell periphery (double arrowhead). Cells have been fixed/permeabilized byMetOH/acetone, labelled with anti-αIIbβ3 complex-mAb (AP-2), biotinylated anti-mouse IgG and Streptavidin-FITC. Bar, 10 µm.Fig. 2. Immunofluorescent labeling of intracellular αIIbβ3 in 12-(S)-HETE-stimulated B16a cells spread on fibronectin. Note a significant shiftin the pattern of labeling; elongated tubulovesicular structures became more evident (arrowhead) especially at the cell periphery (doublearrowhead). Methodology as for Fig. 1. Bar, 10 µm.Fig. 3. Immunoelectron microscopic localization of intracellular αIIbβ3 in fibronectin-adherent B16 cells. Note that perinuclear vesicle (a) aswell as elongated tubule (b) both contains intraluminal gold labeling (arrowheads). Cells were fixed in paraformaldehyde, dehydrated bycryosubstitution using PLT, embedded into Lowicryl HM-20 and sections stained for αIIbβ3 complex using anti-complex mAb AP-2 andGAMG5. Counterstaining was performed by lead citrate. Bar, 0.1 µm.Fig. 4. Immunoelectron microscopic localization of intracellular αIIbβ3 in fibronectin-adherent B16a cells. (a) Electron dense reaction productcan be seen on intraluminal vesicles (arrow) of a multivesicular endosome. (b) negative control. Tumor cells have been fixed inparaformaldehyde/glutaraldehyde mixture, permeabilized with Triton X-100, labelled with anti-αIIbβ3 complex mAb AP-2 and goat anti-mouseIgG-peroxidase conjugate, DAB, then processed for transmission electron microscopy using OsO4. Bar, 0.1 µm.

preformed receptor complexes. In these cells, αIIbβ3 was foundto be associated with vesicles located in a perinuclear area cor-responding to the putative Golgi zone (Fig. 1). The eicosanoid12-(S)-HETE was reported to increase surface expression ofαIIbβ3 on B16a cells with an optimal response at 0.1 µM(Chopra et al., 1991). Stimulation of adherent B16a cells with12-(S)-HETE (0.1 µM, 15 minutes) resulted in a change inlabeling pattern for αIIbβ3 from vesicular to elongated tubuleswhich extended to the cell periphery (Fig. 2). These two struc-tural components were termed the TVS compartment. Next, theintracellular compartment containing αIIbβ3 was studied at theultrastructural level. Ultrathin sections of B16a cells which hadbeen low temperature embedded were labeled with mAb AP-2. Immunogold label was frequently localized to both perinu-clear vesicles (Fig. 3a) as well as to the elongated tubular struc-tures (Fig. 3b). To further confirm these data, intracellularαIIbβ3 was localized using a postfixation immunoperoxidase

labeling technique. In these samples a postive reaction forαIIbβ3 was found on the membranes of cytoplasmic vesicles(data not shown) as well as on inner vesicles of the tubulovesic-ular bodies (Fig. 4a). One of the major cytoplasmictubulovesicular compartments belongs to theendosomes/trans-Golgi network (TGN; Anderson and Pathak,1985; Hopkins, 1986, 1990). Therefore we have used the trans-ferrin/transferrin receptor system as a marker of this compart-ment. Double labeling of intracellular αIIbβ3 (Fig. 5a) and theendosomes for transferrin/transferrin receptors (Fig. 5b)indicated an almost complete colocalization of endosomes andintracellular αIIbβ3 suggesting that the αIIbβ3-TVS poolbelongs to endosomes/TGN system.

Immunocytochemistry at the ultrastructural level was usedto localize αIIbβ3 on the surface of unstimulated B16a cells. ByWMTEM rare microdomains covered by αIIbβ3 were observedin these cells (Fig. 6a). However, in 12-(S)-HETE stimulated

2179Effect of 12-(S)-HETE on αIIbβ3 in melanoma cells

Fig. 5. Immunofluorescent double labeling of αIIbβ3 and intracellular transferrin in non-stimulated fibronectin-spread B16a cells. Note thatαIIbβ3-containing vesicles (a; arrow) and intracellular transferrin (b; arrow) frequently colocalize. Adherent tumor cells were incubated withtransferrin for 15 minutes at 37°C and were fixed/permeabilized with MetOH/acetone, labelled for transferrin (goat anti-transferrin IgG; rabbitanti-goat IgG-biotin and Streptavidin Texas Red) and for a complex (mAb AP-2; biotinylated goat anti-mouse IgG; Streptavidin-FITC). Bar, 10 µm.

cells the increased amount of αIIbβ3 was observed at the plasmamembrane, in particular on microvilli (Fig. 6b). These resultswere confirmed in unstimulated (Fig. 6c) and stimulated (Fig.6d) B16a cells by TEM.

The effect of 12-(S)-HETE on the morphological structureof the TVS and associated cytoskeletal elements was charac-terized also by WMTEM. In unstimulated cells cytoplasmicvesicles and intermediate filaments (as determined by theirdiameter) were found predominantly in the perinuclear region(Fig. 7a and b). Following 12-(S)-HETE stimulation, organelletranslocation to the cell periphery was observed. Unlike theTVS in unstimulated cells, which possessed a predominantlyvesicular appearance, 12-(S)-HETE stimulated cells possesseda TVS which was tubular and elongated (Fig. 7c). In addition,occasional microtubules or intermediate filaments were foundin association with the tubular structures (Fig. 7c,d).

Previously we demonstrated that the surface expression ofαIIbβ3 on suspended B16a cells in response to 12-(S)-HETEwas dependent upon an intact vimentin intermediate filamentnetwork (Chopra et al., 1991). However, adhesion andspreading of B16a cells were also dependent on intact micro-tubules (Tímár et al., 1993). As tumor cells establish a well-developed cytoskeletal network in the adherent state, we deter-mined whether the vesicular to tubular transition of integrincontaining TVS (Fig. 8a versus c) and the increased surfaceexpression of αIIbβ3 following 12-(S)-HETE treatment (Fig. 8bversus d) was dependent upon intact microtubules or interme-diate filaments in matrix-adherent cells. The vimentin inter-mediate filament system was disrupted with acrylamide ornocodazole as previously described (Tímár et al., 1993), andthe cells were stimulated with 12-(S)-HETE and labeled forintracellular and surface αIIbβ3 as described above. Disruptionof both intermediate filaments and microtubules blocked thevesicular to tubular transition of the αIIbβ3 pool (Fig. 8e andg) and prevented the 12-(S)-HETE-induced increase in surfaceexpression (Fig. 8f and h) quantitated in situ with laser fluo-rescence imaging (Fig. 8i).

The role of the Golgi system in the translocation of theαIIbβ3 pool to the cell surface after 12-(S)-HETE stimulationwas studied by using specific disruption of the Golgi by

brefeldin A (Doms et al., 1989; Ferreira et al., 1991; Lippin-cott-Schwartz et al., 1989). Preliminary studies using BFA atvarious concentrations for different time-periods indicated that120 minutes treatment with 10 µg/ml is optimal and non-toxic(data not shown) whereas pretreatment of unstimulated B16acells with brefeldin A alone at a 5 µg/ml dose for 60 minutesinduced a slight vesicular to tubular transition of αIIbβ3vesicles (data not shown). The brefeldin A pretreatment (Fig.9b) (120 minutes; 10 µg/ml) inhibited 12-(S)-HETE inducedelongation of the αIIbβ3 containing TVS (Fig. 9a) whenbrefeldin A pretreated cells (data not shown) in the absence of12-(S)-HETE resembled controls (Fig. 1) in that the αIIbβ3 con-taining TVS was vesicular. 12-(S)-HETE increased surfaceexpression of αIIbβ3 on B16a cells as determined by flowcytometry (Fig. 9c). This response was completely inhibited byappropriate pretreatment with brefeldin A (Fig. 9c).

Previously we have observed that the 12-(S)-HETE effect ismediated by PKC (Tímár et al., 1992, 1993). Therefore, weanalyzed the effect of a selective PKC inhibitor, calphostin C,on the surface expression of αIIbβ3. In control cells the lowlevel of surface expression of αIIbβ3 decreased significantlyafter only 1 µM calphostin C pretreatment for 10 minutes (Fig.10). However, the 12-(S)-HETE induced increase in surfaceintegrin expression was significantly inhibited at a dose rangefrom 0.1-0.5 µM (Fig. 10).

Surface labeling of cells with transferrin or HRP can identifyintracellular organelles involved in endocytosis (Lang anddeChastellier, 1985). In unstimulated and 12-(S)-HETE stim-ulated B16a cells, transferrin (Fig. 5b) and HRP label (data notshown) were localized to TVS structures, suggesting that theyare part of the endosomal compartment. After binding tosoluble ligands integrin receptor-ligand complexes are inter-nalized by endocytosis. Therefore, in order to identify the cyto-plasmic compartment involved in the endocytosis of αIIbβ3,B16a cells were labeled with mAb10E5 (immunofluorescence)or mAbAP-2 (immunogold) at 4°C and then incubated at 37°Cfor 15 or 30 minutes to induce the internalization of thereceptor-ligand complex. On the surface of B16a cells, αIIbβ3was localized to areas of high and low receptor density (Fig.11a) as previously described (Chopra et al., 1991; Tímár et al.,

2180 J. Tímár and others

Fig. 6. Surface localisation of αIIbβ3 on fibronectin-adherent unstimulated B16a cells. (a) Immuno-WMTEM. Note the rare presence of gold-label on the apical surface. Fixation: 1% paraformaledehyde/0.1% glutaraldehyde; mAb AP-2, biotinylated goat anti-mouse IgG, Streptavidin-G15. Bar, 0.1 µm. (b) Immuno-TEM. Apical surface membrane as well as surface-process contains microdomains (G5 particles; arrows)positive for αIIbβ3. Fixation: as in case of (a); incubations, mAb AP-2, GAMG5. Postfixation: using OsO4. Bar, 0.1 µm. Surface localization ofαIIbβ3 on fibronectin adherent 12-(S)-HETE-stimulated B16a cells. (c) Immuno-WMTEM. Note the accumulation of gold-label on surfaceprojections. Fixation and labeling as in case of a. (d) Immuno-TEM. Surface membrane as well as membrane elaboration frequently containgold-label (arrows). Fixation and labeling as in case of b. Bar, 0.1 µm.

1993). After 15 minutes incubation at 37°C the label waslocalized predominantly to tubular structures as determined byimmunofluorescence (Fig. 11b) and WMTEM (Fig. 11c).However, following 30 minutes incubation the label waslocalized predominantly in vesicular structures (Fig. 11e)located in the perinuclear area of the cell (Fig. 11d). Parallelinternalization of transferrin and surface αIIbβ3 (after ligandbinding) was also performed to study the relation of internal-ized integrin to endosomes. Our data indicated that after 15minutes incubation at 37°C the internalized integrin (Fig. 12a)was localized to similar TVS structures as transferrin (Fig. 12band c) indicating that the cytoplasmic compartment involvedin the internalization of both ligands most probably belongs tothe endosome/TGN network.

DISCUSSION

The binding of soluble ligands to membrane receptors induces

internalization of the ligand-receptor complex into a dynamicintracellular compartment called endosomes (Hopkins, 1986).The endosomal compartment is connected to the trans-Golginetwork (TGN), the latter providing a link between endocy-totic and exocytotic pathways for the anterograde and retro-grade translocation of receptors (Anderson and Pathak, 1985).The TGN compartment has a tubulovesicular morphologywhich can be altered in response to a variety of extracellularand intracellular signals (Hopkins et al., 1990).

The intracellular pool of some matrix receptors, i.e. α5β1,αvβ3, αIIbβ3 and laminin receptors, exist in the form of cyto-plasmic granules termed adhesomes (Sczekan and Juliano,1990; Wenzel-Drake et al., 1986; Wenzel-Drake, 1990). Onefunction of this intracellular integrin pool is to accumulateinternalized integrin-ligand complexes (Raub and Kuentzel,1989; Sczekan and Juliano, 1990). In addition, this compart-ment serves as a pool of integrin receptors which may translo-cate to the plasma membrane as part of a recycling loop(Bretscher, 1989; Molnar et al., 1987; Wenzel-Drake, 1990).

2181Effect of 12-(S)-HETE on αIIbβ3 in melanoma cells

Fig. 7. WMTEM of tubulovesicular structures in adherent unstimulated B16a cells. (a) The structure appears to be predominantly vesicular (V)surrounded by a loose network of thin filaments (F), but (b) some tubular profiles (T) were occasionally surrounded by microtubules (M). (c)Intermediate filaments (arrowheads). Appearance of cytoplasmic organelles in 12-(S)-HETE-stimulated B16a cells (c,d); note the elongatedtubulovesicular structures (c, arrowheads) surrounded by microtubules (M). High power view WMTEM picture demonstrating tubulovesicularstructures in 12-(S)-HETE-stimulated B16a cells indicates that these vesicles are also surrounded by an intermediate filament network (d,arrowheads). Bar, 0.1 µm.

In the present study we characterized a dynamic intracellu-lar pool of the αIIbβ3 integrin, a multifunctional receptor forfibrinogen, fibronectin, vitronectin, vonWillebrand factor,thrombospondin and osteopontin (Honn and Tang, 1992).αIIbβ3 was previously believed to be restricted to cells ofmegakaryocytic lineage (Albeda and Buck, 1990). RecentlyαIIb and β3 were identified in B16a cells by immunoblotting(Chopra et al., 1991), immunoprecipitation, Southern andnorthern blotting, in addition to reverse transcriptase poly-merase chain reaction (RT-PCR) and sequence analysis(Chang et al., 1992; Chen et al., 1992). In fact, αIIbβ3 is widelydistributed among human and rodent tumors (Chen et al.,1992). The message level and plasma membrane expression ofαIIbβ3 correlates positively with the metastatic potential of sub-populations of B16a cells (Chang et al., 1992; Honn and Tang,1992) and this receptor is demonstrated to mediate tumor cell-platelet (Grossi et al., 1988; Chopra et al., 1992), and tumorcell-endothelial cell (Honn and Tang, 1992; Tang et al.,1993a,b) interactions. In addition both αIIbβ3 mediates 12-(S)-

HETE stimulated B16a cell spreading on fibronectin (Tímár etal., 1992). Therefore this receptor may play a significant rolein the metastatic process and hence the elucidation of themechanism(s) responsible for its plasma membrane expressionand the regulation of those mechanisms is of importance.

In this study we provide evidence that tumor cell αIIbβ3,similar to other integrins, is stored in cytoplasmic vesicles inunstimulated cells. In B16a cells this receptor pool appears astubulovesicular structures which morphologically resembleendosomes (Hopkins et al., 1990). Colocalization of anendosomal marker, transferrin/transferrin-receptor and theintracellular αIIbβ3, strongly support this hypothesis.Endosomes contain newly synthesized receptors as well asserving as a depository for internalized receptors. When B16acells were labeled with αIIbβ3 complex specific monoclonalantibodies at 4°C and then incubated at 37°C to allow receptorinternalization, the label expressed first in tubular structuresnear the cell periphery and later in vesicular structures locatedin the perinuclear area. This progressive retrograde localiza-

2182 J. Tímár and others

Fig. 8. Effect of cytoskeleton disruption on intracellular integrin pool and on thesurface receptor expression. (a) Intracellular appearance of αIIbβ3 in unstimulatedadherent B16a cells. Note the predominant vesicular forms of TVS.Immunofluorescence as in Fig. 1. Bar, 10 µm. (b) Surface localization of αIIbβ3 onunstimulated adherent B16a cells detected by immunofluorescence performed after 2%paraformaldehyde fixation. Digitalized low power laser fluorescence image; wherespecified color corresponds to signal intensity. (c) Effect of 12-(S)-HETE (15 minutes,0.1 µM) on the intracellular αIIbβ3 pool detected with immunofluorescence as in caseof a. Note the predominant tubular shape of the cytoplasmic pool. Bar, 10 µm. (d)Effect of 12-(S)-HETE on the surface expression of αIIbβ3 detected byimmunofluorescence after 2% paraformaldehyde fixation. Digitalized low power laserfluorescence image (as in case of b). Note the increased surface labeling. (e) Effect ofnocodazole pretreatment (1 hour, 3 µg/ml) on 12-(S)-HETE induced alterations in the

intracellular αIIbβ3 pool detected as in case of Fig. 2. Note the predominant vesicular shape of the compartment situated in the perinuclearregion. Bar, 10 µm. (f) Effect of nocodazole pretreatment on the 12-(S)-HETE-induced surface expression of αIIbβ3 detected byimmunofluorescence and digitalized laser fluorescence. Note the low level of receptor expression at the cell surface compared to d. (g) Effect ofacrylamide pretreatment (4 hours, 10 mM) on the 12-(S)-HETE-induced alterations in the intracellular pool of αIIbβ3 detected byimmunofluorescence. Note the predominant perinuclear vesicular labeling. Bar, 10 µm. (h) Effect of acrylamide pretreatment on the 12-(S)-HETE-induced surface expression of αIIbβ3 detected by immunofluorescence and digitalized laser fluorescence. Note the low level offluorescence at the cell surface compared to d. (i) Effect of cytoskeleton disruptions on the surface expression of αIIbβ3 measured by digitalizedlaser fluorescence imaging. Four selected areas were measured for fluorescence intensity. At each point a minimum of 200 cells were measuredand data expressed as mean ± s.d.

2183Effect of 12-(S)-HETE on αIIbβ3 in melanoma cells

Fig. 9. Intracellular localisation of αIIbβ3 in B16 cellsby immunofluorescence. (a) 12-(S)-HETE stimulatedadherent B16a cells. Note the elongated tubularstructures in the cytoplasm (arrowheads). (b) 12-(S)-HETE stimulated adherent B16a cell after 2 hourpretreatment with brefeldin A (10 µg/ml).Cytoplasmic αIIbβ3 appears to be associated with apredominantly vesicular perinuclear compartment(arrowheads). Methodology: as in case of Fig. 1. Bar,10 µm. (c) Flow cytometric measurement of surfaceαIIbβ3 on suspended B16a cells. All treatments wereperformed on suspended cells. CTR, non-treated cellskept in serum-free MEM for 120+15 minutes at 37°C;CTR/BFA, brefeldin A pretreatment for 120 minutes(10 µg/ml) + 15 minutes at 37°C; 12-(S)-HETE, 120minutes in MEM at 37°C, 15 minutes stimulation with0.1 µM 12-(S)-HETE; BFA/HETE, brefeldin Apretreatment (10 µg/ml) for 120 minutes +15 minutes12-(S)-HETE-stimulation. Data are expressed as meanrelative fluorescence ± s.d., n=3. Fixation andimmunolabeling: as in case of Fig. 10a.

tion of the receptor resembles, in reverse, the anterograde dis-persion of αIIbβ3 containing vesicles into tubular structuresafter 12-(S)-HETE stimulation. Colocalization of the internal-ized transferrin and αIIbβ3 into similar TVS profiles suggeststhat these structures are related to the endosome/TGN network.Collectively, these results also suggest a dynamic pool ofαIIbβ3 receptor which may cycle between endosomes (i.e.TVS) and the plasma membrane.

In platelets αIIbβ3 upregulation is directed by ligand con-centration or surface activation, where the receptor redistrib-utes at the plasma membrane to the site of cellular or matrixinteractions (Lewis et al., 1990; Wenzel-Drake, 1990; Whiteand Escolar, 1990). In melanoma cells αIIbβ3 can be upregu-lated at the cell surface by treatment of cells with 12-(S)-HETE, a stimulator of PKC activity (Liu et al., 1991), whichinduces accumulation of the integrin at filo- and lamellopodia

Fig. 10. Effect of calphostin C pretreatment on the surfaceexpression of αIIbβ3 (flow cytometry). Suspended tumor cells werepretreated with light-activated calphostin C for 10 minutes at 37°C(diluted in serum-free MEM). These cells were treated with 0.1 µM12-(S)-HETE for 15 minutes and labelled for surface αIIbβ3 as incase of Fig. 9. Expression measured by flow cytometry in triplicatesamples and expressed as % mean fluorescence ± s.d. where controlis 100.

as well as in focal adhesion plaques (Chopra et al., 1991; Tímáret al., 1992). The appearence of αIIbβ3 at specific membraneareas after PKC stimulation induced by 12-(S)-HETE suggestsa polarized translocation of integrin receptor from its intracel-lular TVS pool.

2184 J. Tímár and others

Fig. 11. Internalization of surfaceαIIbβ3 in B16a cells. (a) Immunofluorescencelocalization of αIIbβ3 at the apicalmembrane of an unstimulatedadherent B16a cell. Note therandom distribution of discretereceptor-positive microdomains.4°C labeling: mAb 10E5,biotinylated goat anti-mouse IgG,Streptavidin-FITC. Fixation: 2%paraformaldehyde. Bar, 10 µm.(b) Internalisation of αIIbβ3 intoadherent B16 cell for 15 minutes.Cytoplasmic elongatedtubulovesicular structures(arrowheads) are frequent on thecytoplasm irradiating from theperinuclear zone.Immunofluorescence: mAb 10E5was incubated with cells for 15minutes at 37°C, cells have beenfixed with MetOH/acetone, thenwere labelled with biotinylatedgoat anti-mouse IgG andStreptavidin-FITC. Bar, 10 µm.(c) 15 minutes internalisation ofαIIbβ3 into non-stimulated B16acell. Immuno-WMTEM. Note thepresence of gold-label inelongated cytoplasmic tubule(arrowhead). Adherent tumorcells have been labelled withmAb AP-2, biotinylated goat anti-mouse IgG and Streptavidin-G15at 4°C, incubated at 37°C thenwhere fixed and processed as incase of Fig. 6. Bar, 0.1 µm. (d)Internalisation of αIIbβ3 intoadherent B16 cell for 30 minutes.Note the clear vesicularappearence of αIIbβ3-containingcytoplasmic structuresaccumulating around the nucleus(arrowheads).Immunofluorescence: mAb 10E5was incubated with cells for 30minutes at 37°C, which wasdetected as in case of b. Bar, 10

µm. (e) 15 minutes internalisation of αIIbβ3 into 12-(S)-HETE-stimulated B16a cell. Immuno-WMTEM. Note the presence of gold label invesicular structure (arrowhead). Methodology as in case of Fig. 6. Bar, 0.1 µm.

The αIIbβ3 integrin-containing TVS in B16a cells may be partof the Golgi/trans-Golgi network. Brefeldin A reversiblyinhibits intracellular membrane transport from the endoplasmicreticulum to the Golgi (Doms et al., 1989; Lippincott-Schwartzet al., 1989) acting at the cis-intermediate Golgi interface. Thisallows the TGN to be functionally dissected from the remainderof the Golgi complex (Ladinsky and Howell, 1992). Also,brefeldin A reversibly inhibits intracellular membrane transportfrom the endoplasmic reticulum to the Golgi (Doms et al., 1989;Lippincott-Schwartz et al., 1989). Pretreatment of B16a cells

with brefeldin A inhibited the 12-(S)-HETE-induced vesicularto tubular transition of the integrin-containing TVS as well asthe immediate upregulation of the integrin at the cell surface.These data suggest that the TVS/endosomes containing αIIbβ3are connected to the TGN of the Golgi system.

The cytoskeletal association of endosomes and TGN is notknown yet. In B16a cells we found that TVS as well as theintegrin-containing TVS (data not shown) in unstimulated stateare surrounded by vimentin intermediate filaments but rarelywith microtubules. After PKC stimulation with 12-(S)-HETE,

2185Effect of 12-(S)-HETE on αIIbβ3 in melanoma cells

Fig. 12. Parallel internalization of transferrin and ligand-bound αIIbβ3 in adherent B16a cells. Tumor cells were incubated with transferrin andmAb-AP-2 for 30 minutes at 4°C, washed and incubated at 37°C for 15 minutes. The internalized transferrin was developed as in case of Fig.5b (using Streptavidin-Texas Red) while the intracellular αIIbβ3-AP2 complex was labelled with anti-mouse IgG-biotin and Streptavidin-FITC.Digitalized FITC (a) and Texas Red (b) pictures of the same cell were superimposed by using Photostyler software (Aldus) (c). The majority ofintracellular TVS structures in a and b are colocalized (c). Bar, 10 µm.

αIIbβ3 αIIbβ3+Transferrin

receptor

Transferrinreceptor

TVS became associated frequently with microtubules and inter-mediate filaments (Tang et al., 1993a,b; Tímár et al., 1993). Thefunctional significance of this was further supported by theobservation that disruption of microtubules or intermediatefilaments prevents both the vesicular to tubular transition ofTVS as well as the increased surface expression of αIIbβ3induced by 12-(S)-HETE. The organelle translocations alongcytoskeletal filaments involve the function of motor molecules.There are two well-characterized translocation models to date;the microtubules where the mechanochemical motors arekinesin (anterograde) and dynein (retrograde) and the microfil-aments where myosins serve as motor molecules (Hoffman,1992; Taylor, 1992). Recently, kinesin was found to be associ-ated with intermediate filaments in fibroblasts, suggesting thatthis motor molecule may use intermediate filaments as tracks,similar to microtubules, during organelle translocations(Gyoeva and Gelfand, 1991). Our preliminary data indicate thatin B16a cells both kinesin and myosin light chains are associ-ated with TVS (J. Tímár and K. V. Honn, unpublished obser-vations) indicating that TVS translocation in B16a cells alsoinvolve the functions of mechanochemical motors using micro-tubules and intermediate filaments as tracks. Phosphorylationof either the track proteins or the motor molecules is a mainregulator of this complex mechanism (Wang, 1991) and at leastone kinase, PKC, is involved in this phosphorylation response(Ciesielski-Treska, 1991; Grant and Aunis, 1990). Recentstudies on B16a cells indicated that cytoskeletal proteins,vimentin and MLC, are heavily phosphorylated by PKC after

stimulation with 12-(S)-HETE, inducing a complex rearrange-ment of both the microtubules as well as the intermediatefilament systems (Tang et al., 1993a,b; Tímár et al., 1993) sug-gesting a role for PKC in regulation of cytoskeletal functionssuch as organelle translocation. These ideas were furthersupported by our finding that both basal as well as the 12-(S)-HETE-stimulated surface expression of αIIbβ3 integrin issensitive to a selective PKC inhibitor, calphostin C. Thepolarized translocation of αIIbβ3 receptor by TVS in B16a cellsafter 12-(S)-HETE stimulation is mediated by microtubules andintermediate filaments (Chopra et al., 1991; Tímár et al., 1992)and is most probably involved in the function of well knownmechanochemical molecules such as kinesin and/or MLC.Further studies are underway to clarify these points.

This work was supported by NIH grants CA-47115-04 and CA-29997-08 (K.V.H.), FIRCA TW 285-01 (K.V.H. and J.T.), by NATOLinkage Grant Programme LG 921311 (K.V.H. and J.T.), and by theHungarian National Science Foundation T 6336 (J.T.).

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(Received 17 June 1994 - Accepted, in revised form, 16 December 1994)