Molecular proximity of seprase and the urokinase-type plasminogen activator receptor on malignant melanoma cell membranes: dependence on ß1 integrins and the cytoskeleton
Vira V. Artym1,
Andrei L. Kindzelskii1,
Wen-Tien Chen2 and
Howard R. Petty1,3
1 Department of Biological Sciences, Wayne State University, Detroit, MI 48202 and
2 Department of Medicine, Division of Medical Oncology, State University of New York, Stony Brook, NY 11794-8160, USA
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Abstract
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Previous studies have shown that several proteolytic enzymes are associated with membrane protrusions at the leading edge of migrating tumor cells. In this study we demonstrate that seprase and the urokinase plasminogen activator receptor (uPAR), co-localize in the plasma membrane of LOX malignant melanoma cells. Cells were labeled with fluorochrome-conjugated monoclonal antibodies (mAb) directed against seprase and uPAR. Proximity between these two molecules was detected with resonance energy transfer (RET) imaging, single-cell emission spectrophotometry, and single-cell excitation spectrophotometry. Significant RET signals were detected on LOX cells when adherent to uncoated and extracellular matrix (ECM)-coated surfaces. This indicates that seprase and uPAR are within
7 nm in the plasma membrane of LOX cells. When LOX cells adhered to a 3D extracellular-like matrix, sepraseuPAR complexes were found to be associated with invadopodia. Further microscopy experiments demonstrated gelatinolytic activity, a functional attribute of seprase, in association with sepraseuPAR membrane domains. Formation of sepraseuPAR membrane complexes is dependent upon both the cytoskeleton and integrins. Specifically, the involvement of ß1-integrins was demonstrated by the inhibition of RET by an inhibitory anti-ß1-integrin mAb. Based on these findings, we speculate that formation of heterogeneous lytic domains in the invading membranes of LOX cells increases the efficiency of directed pericellular proteolysis.
Abbreviations: AMCA, 7-amino-4-methylcoumarin-3-acetic acid; ECM, extracellular matrix; FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; RET, resonance energy transfer; TRITC, tetramethylrhodamine isothiocyanate; uPAR, urokinase plasminogen activator receptor
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Introduction
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The metastatic potential of aggressive tumor cells is dependent upon the localization of plasma membrane-bound proteolytic systems at sites of cell invasion (1). These enzymes provide the directed pericellular proteolysis of the extracellular matrix (ECM) necessary for tumor invasiveness and metastasis (2). ECMs consist of several components including collagens, proteoglycans and glycosaminoglycans. Some ECM molecules can be degraded by a variety of enzymes whereas the proteolysis of others requires specific enzymes. Therefore, a range of proteolytic enzymes may be associated with the leading edge of migrating tumor cells (24).
Several mechanisms can contribute to the formation of membrane domains. One mechanism thought to promote the formation of protein domains in cell membranes is glycosphingolipid-enriched regions. Another mechanism promoting the non-random co-distribution of membrane proteins is lateral interaction between membrane proteins or between membrane and cytoskeletal proteins. In addition, cytoskeletal barriers can also act as corrals to limit the mixing of membrane molecules. One approach to characterizing membrane protein domains is the analysis of the molecular proximity of their components. The molecular proximity and of membrane components has been studied by a variety of methods including immuno-precipitation, co-fractionation, chemical cross-linking, fluorescence microscopy and resonance energy transfer (RET). Fluorescence microscopy and RET methods possess the advantage that they can be used on living cells, thereby avoiding potential complications due to cell disruption and detergent solubilization (5). Although fluorescence microscopy can be used to co-localize two molecules on a membrane, the resolution is limited to that of the Rayleigh criterion (
200 nm). On the other hand, RET can be used to detect molecular proximity of fluorescent membrane labels separated by
7 nm. As this distance is approximately the diameter of many multi-pass membrane proteins, positive RET signals suggest the pair of labeled molecules are nearest neighbors. For example, RET has been demonstrated for several membrane components including: concanavalin A receptors of normal and transformed fibroblasts (6), insulin receptors (7), several lymphocyte receptors (8), ß2 integrins and urokinase plasminogen activator receptor (uPAR) on neutrophils (9), and ß1, ß3 integrins with uPAR on fibrosacoma cells (10). These receptors form supramolecular receptor structures that are significant in transmembrane signaling, cellular immunology and cell migration.
Here we demonstrate the formation of supramolecular complexes in the plasma membranes of LOX melanoma cells involving two protease systems: seprase and urokinase-type plasminogen activator. These enzymes are associated with highly metastatic tumors (1116). Seprase, a 170 kDa transmembrane protein, degrades gelatin but not laminin, fibronectin, fibrin and casein (17). The uPA system includes serine proteinases uPA, plasmin and the uPAR, which is a GPI-linked membrane protein (18). uPA converts the tissue zymogen plasminogen into the plasmin, a serine proteinase with broad substrate specificity. We demonstrate lateral proximity of seprase and uPAR, thus suggesting the formation of proteinase-rich membrane domains, which may contribute to the ECM degradation at the cellular invasion front and the metastatic ability of cells.
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Materials and methods
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Materials
Fibronectin and laminin were purchased from Life Technologies (Grand Island, NY); vitronectin was bought from Boehringer Mannheim (Indianapolis, IN). Fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC) and Alexa Fluor 546 for antibody conjugation as well as DQ gelatin were obtained from Molecular Probes, (Eugene, OR). 1,10-Phenanthroline monohydrate was purchased from Fluka Chemika (Ronkonkoma, NY). American Diagnostics Corporation (Joplin, MO) provided plasminogen activator inhibitor 1 (PAI-1). Colchicine and cytochalasin B were purchased from Sigma Chemical Co. (St Louis, MO).
Antibodies
Rat monoclonal D28 antibodies of IgG2a isotype were prepared as described (17). Rat monoclonal C27 antibodies of IgG2a isotype, identified as inhibitory anti-ß1 integrin antibodies, were prepared as described (1,19). Mouse monoclonal anti-uPAR F(ab)2 antibody fragments of IgG2a isotype were kindly provided by Dr Robert F. Todd III (Department of Internal Medicine, University of Michigan School of Medicine, Ann Arbor, MI).
Monoclonal anti-seprase D28, and anti-uPAR antibodies were conjugated with FITC and TRITC, respectively. The pH of antibody solutions was increased by overnight dialysis against carbonate buffer (pH 9.6; 0.2 M). The solutions were then mixed with TRITC or FITC at a molar ratio 1:13 for 3 h with shaking followed by overnight dialysis against PBS (pH change from 9.6 to 7.4) and Sephadex G25 chromatography.
Cell culture
The human amelanotic malignant melanoma cell line LOX was kindly provided by Dr Oystein Fodstad (Department of Tumor Biology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo, Norway). LOX cells were cultured in Dulbeccos modified Eagle medium and RPMI-1640 medium (Life Technologies) at 1:1 ratio supplemented with 10% heat-inactivated fetal calf serum (Life Technologies), 5% Nu-serum IV (Becton Dickinson, Bedford, MA) and 0.01% antibiotic-antimicotic (Life Technologies) in an atmosphere of 5% CO2 at 37°C.
Indirect immunofluorescence staining
To study seprase and uPAR cell surface localization, LOX tumor cells were incubated for 2 h on variously coated glass coverslips. Coverslips were coated with fibronectin (10 µg/ml), vitronectin (5 µg/ml) or laminin (10 µg/ml) in PBS (pH 7.2) by incubation for 2 h at 37°C or overnight at 4°C, followed by extensive washing. Cells attached to the coverslip were fixed with 3% paraformaldehyde for 15 min at room temperature. Coverslips with attached cells were washed several times with HBSS. Then cells were labeled with monoclonal FITC-conjugated anti-seprase mAb (10 µg/ml) for 1 h at room temperature. After several washes cells were again fixed with 3% paraformaldehyde and blocked with 3% BSA in HBSS, followed by labeling with TRITC-conjugated F(ab)2 fragments of anti-uPAR mAb for 1 h at room temperature. After extensive washing the coverslip was inverted and mounted on a slide. The stained cells were observed using fluorescence microscopy.
Microscopic assessment of gelatinolytic activity
To localize and evaluate gelatinolytic activity, the enzymatic activity of seprase, DQ gelatin was used as a substrate. DQ gelatin is a fluorescein conjugate (gelatin so heavily labeled with fluorescein that fluorescence is quenched). Proteolysis by gelatinase yields fluorescent gelatinFITC peptides. Therefore, the localization of fluorescence indicates the sites of gelatinolytic activity. For this purpose LOX cells were incubated for 3 h on coverslips coated with fluorescein conjugate-gelatin matrix by incubation for 2 h at 37°C. The matrix included: DQ gelatin (0.2%), collagen III (2%) and fibronectin (10 µg/ml). PAI-1 (0.05 mg/ml) or PAI-1 plus 1,10-phenanthroline monohydrate (10 mM) were added to the matrix for inhibitor tests. Extensive washing with HBSS followed cell incubation. Cells attached to the coverslip were fixed with 3% paraformaldehyde for 15 min at room temperature, washed (three times), labeled with anti-seprase TRITC-conjugated antibodies, washed again, then examined using fluorescence microscopy.
Microscopic assessment of invadopodia formation
To demonstrate the formation of invadopodia, a modification of previous methods (19) was employed. As invadopodia are formed parallel to the optical axis of the microscope, we reconstructed 3D images of the gel beneath the tumor cells. The matrix was prepared from 3% collagen, 2% high melting point gelatin, 10 µg/ml fibronectin and 50 µg/ml 7-amino-4-methylcoumarin-3-acetic acid (AMCA). A thin layer of this material was applied to a coverslip then allowed to solidify at room temperature, although it possessed a semi-solid tissue-like consistency at 37°C. The matrix was not allowed to dry. Cells were labeled in suspension with FITCanti-seprase and TRITCanti-uPAR. Cells were washed several times with buffer. A drop of the cell suspension was then placed on the matrix. The sample was placed on a slide and sealed with vacuum grease to prevent evaporation. Samples were incubated for 1 h at 37°C. After collection of FITC, TRITC and RET micrographs (see below), a 3D image of the AMCA label within the matrix was prepared. Images of AMCA fluorescence were collected from multiple focal planes around and below the cells. To create the images shown below, the pixel intensities were inverted then image stacks were assembled using Microtome software (Vay-Tek, Fairfield, IA;20). Vox-Blast software was used to rotate the 3D image to create the appearance of the invadopodia from the gelatin side of the sample.
Fluorescence microscopy
Cells were observed using an axiovert fluorescence microscope (Carl Zeiss, New York, NY) with mercury illumination interfaced to a computer using Scion image processing software (21). A narrow band-pass discriminating filter set (Omega Optical, Brattleboro, VT) was used with excitation at 485/22 nm and emission at 530/30 nm for FITC and an excitation of 540/20 nm and emission at 590/30 nm for TRITC. For AMCA, excitation was provided by a 380/15 and a 450/30 emission filter was used. Long-pass dichroic mirrors of 400, 510 and 560 nm were used for AMCA, FITC and TRITC, respectively. For RET imaging, a 485/22 nm narrow band-pass discriminating filter was used for excitation and a 590/30 nm filter was used for emission. The fluorescence images were collected with a Peltier-cooled intensified charge-coupled device camera (Geniisys; Dage-MTI, Michigan City, IN). DIC photomicrographs were taken using Zeiss polarizers and a charge-coupled device camera (Model 72; Dage-MTI).
Single cell emission spectrophotometry
Energy transfer was also examined by means of microscope spectrophotometer apparatus (21,22). Fluorescence emission spectra were collected from single cells by a Peltier-cooled IMAX camera with a liquid nitrogen cooled intensifier (Princeton Instruments, Princeton, NJ) attached to modified Zeiss axiovert fluorescence microscope (Carl Zeiss). Winspec software (Princeton Instruments) was used to analyze spectrophotometric data. For RET image observation, a 485/22 nm narrow band-pass discriminating filter was used for excitation; long-pass dichroic mirror and a 520 nm long-pass filter were used for emission. RET intensity levels were obtained by calculating the difference between the intensity of the samples and that of the background. Intensity levels are given as the mean ± SE. P values were calculated using Microsoft Excel 2000 software.
Excitation spectrophotometry
RET was also detected by excitation spectrophotometry (Photon Technology International, Lawrenceville, NJ). A monochromator and a fiber-optically coupled xenon lamp were controlled by FeliX software (Photon Technology International). Excitation spectra were collected from single cells by a cooled PMT system (Photon Technology International) attached to Zeiss axiovert 35 (Carl Zeiss) fluorescence microscope. For Alexa-546 and RET spectrophotometry 570 nm long-pass dichroic mirror and 565 nm long-pass emission filter were used. For FITC spectrophotometry, 505 nm long-pass dichroic mirror and 520 nm long-pass emission filter were used. Excitation spectra were processed by FeliX software.
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Results
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To study the molecular proximity of distinct membrane proteases on tumor cells, we employed the LOX malignant melanoma cell line. The expression of uPAR (CD87) and seprase on LOX cells were demonstrated previously by immunocytochemistry (23) and immunofluorescence microscopy with D28 monoclonal rat antibody (13), respectively.
Immunofluorescence microscopy of uPAR and seprase on the plasma membrane of LOX cells
Based on previous studies showing uPAR to integrin proximity on the lamellipodia of neutrophils (9,24) and tumor cells (10) and seprase to integrin association on LOX cell invadopodia (1), we hypothesized that uPAR and seprase may co-localize on cell membranes. An immunofluorescence microscope was employed to study uPAR and seprase distribution on LOX cell membranes. LOX cells were cultured on untreated glass coverslips and coverslips coated with fibronectin, laminin or vitronectin for 2 h. The presence of ECM substrates resulted in enhanced cell adherence and polarization, thus facilitating experimentation. Cell membrane-associated uPAR and seprase were labeled with F(ab)2 fragments of anti-uPAR TRITC-conjugated mAb and anti-seprase FITC-conjugated D28 mAb, respectively. Immunofluorescence labeling of uPAR and seprase revealed a non-uniform distribution of these molecules on LOX cell membranes (Figure 1
). uPAR and seprase were found on the membranes of LOX cells adherent to the unmodified glass and ECM-coated substrates. Anti-seprase labeling was generally concentrated into smaller regions of LOX cell plasma membranes while adherent to fibronectin and laminin. Because the formation sepraseuPAR enriched domains on LOX cells adherent to vitronectin for 2h was seen in a low percentage of cells (data not shown), we speculate that their formation requires a longer period of time during adherence to this substrate. In general, seprase distribution patterns on cells adherent to glass and ECM substrates resembled those of uPAR and the labeling patterns overlapped.

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Fig. 1. Co-localization and RET of seprase and uPAR on LOX cells attached to different substrates. LOX cells attached to glass, fibronectin-, laminin- or vitronectin-coated coverslips were examined by immunofluorescence microscopy. Cells were labeled with FITC-conjugated anti-seprase D28 mAb (B, F, J and N) and TRITC-conjugated anti-uPAR mAb (C, G, K and O). Columns 14: DIC, fluorescence of anti-seprase, fluorescence of anti-uPAR and RET. Rows 14: glass, fibronectin, laminin and vitronectin. Immunofluorescence and RET data were collected from four to six independent trials for each substrate. During each trial 3040 cells were examined (x600).
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Detection of uPAR and seprase physical proximity on LOX cells by RET imaging
Although superposition of the labeling patterns demonstrates that uPAR and seprase collect in the same vicinity, it does not show molecular proximity. Therefore, RET microscopy was employed to examine the molecular proximity of uPAR and seprase on LOX cells adherent to glass and ECM substrates (fibronectin, laminin, vitronectin). Figure 1
(column 4) illustrates the RET between labeled seprase and uPAR membrane proteins. RET between uPAR and seprase molecules suggests that these molecules are within
7 nm of one another and, therefore, within molecular proximity on the cell membrane (25). Thus, seprase-to-uPAR RET indicates the formation of sepraseuPAR supramolecular complexes on LOX cell membranes, which may be punctate or diffuse in nature.
SepraseuPAR complexes are found at lamellipodia
Inasmuch as seprase preferentially localizes to invading protrusions of the cell surface (invadopodia) of LOX cells adherent to ECM components (13), we hypothesized that sepraseuPAR complexes are located on invadopodia. To test this idea, LOX cells were labeled with TRITC-conjugated anti-uPAR and FITC-conjugated anti-seprase, as described above, then placed on matrices composed of high melting point gelatin, collagen, fibronectin and AMCA. DIC, FITC, TRITC and RET micrographs of the cells were collected as described above. Images of the matrix were also collected by exciting the matrix label AMCA. AMCA micrographs at multiple focal places were collected followed by 3D computer deconvolution to remove out-of-focus AMCA fluorescence. Using this approach, deformation in the matrix due to the overlying tumor cell could be visualized. Figure 2A
D recapitulate some of the data shown above in that LOX cells on another type of substrate also express sepraseuPAR complexes on the cell surface. Figure 2E
shows an image of the matrix looking toward the adherent cell. In this micrograph, five invadopodia are apparent at the corners of the cell, which correspond to sites of RET. Some broad deformation of the matrix is also noted at the center of the cell, which was probably caused by the settling of the dense nucleus of the cell. Thus, invadopodia are sites of sepraseuPAR clusters.

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Fig. 2. Sites of RET correspond to invadopodia regions. LOX cells were labeled with FITC-conjugated anti-seprase D28 mAb and TRITC-conjugated anti-uPAR mAb. Cells were attached to matrices composed of high melting point gelatin, collagen, fibronectin and AMCA. (AD) DIC, fluorescence of anti-seprase, fluorescence of anti-uPAR and RET images, respectively. (E) A 3D image of the matrix below the tumor cell viewed from the perspective of the matrix. Narrow spikes in the matrix correspond to sites of invadopodia extension. By comparing (D) and (E), one can see that sites of RET correspond to the invadopodia. This experiment was repeated on three separate occasions (x700).
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Single cell emission spectrophotometry
Spectrophotometry provides another means of detecting RET. Using quantitative single cell emission spectrophotometry, we examined LOX cells adherent to glass and substrate-coated coverslips. RET was detected between FITC-labeled anti-seprase (donor) and TRITC-labeled anti-uPAR (acceptor) mAbs on LOX cells attached to all of these substrates (Figure 3
). The appearance of an emission peak at
580 nm (Figure 3C
F) in comparison with FITC alone (Figure 3A
) is indicative of energy transfer to the acceptor. Also indicative of RET is the loss of emission intensity of FITC at
535 nm whose peak intensity drops from
16 000 to
10 000 counts.

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Fig. 3. Representative emission spectra of FITC-labeled seprase, TRITC-labeled uPAR, and RET from FITC-labeled seprase to TRITC-labeled uPAR on LOX cells adherent to different substrates. The ordinate and abscissa are the intensity (counts) and wavelength (nm), respectively. (A) Seprase of LOX cell is detected by labeling with D28-FITC-conjugated antibody. (B) uPAR is detected on LOX cells by labeling with anti-uPARTRITC-conjugated mAb. (C) LOX cells attached to glass coverslip labeled with both reagents is shown. In (DF) the substrates were coated with fibronectin, laminin and vitronectin, respectively. RET spectroscopy data were collected from three independent trials for each substrate. During each trial 5060 cells were examined.
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As RET is sensitive to the relative densities of donor and acceptor chromophores (23), the relative heights of donor and acceptor picks should vary according to the donor: acceptor ratio. To confirm and extend our RET results, RET experiments were performed using different donor:acceptor ratios. Representative single cell emission spectrophotometry results for LOX cells adherent to fibronectin-coated coverslips are shown in Figure 4
. An increase in the acceptor concentration resulted in an increase in acceptor peak height. When the donor:acceptor ratio reached 1:10, it was apparent that the higher number of acceptor molecules led to significant quenching of donor fluorescence.

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Fig. 4. RET experiments using different donor:acceptor concentration ratios is shown. RET from FITC-labeled seprase (donor) to TRITC-labeled uPAR (acceptor) was measured by single cell imaging spectrophotometry on LOX cells adherent to the fibronectin-coated coverslips. Donor to acceptor concentration ratios: 1:4 (A), 1:6 (B), 1:8 (C) and 1:10 (D).
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RET between FITC-labeled anti-seprase and TRITC-labeled anti-uPAR mAbs was measured quantitatively by single cell imaging spectrophotometry. Table I
shows that the average RET intensity level was indistinguishable among cells attached to untreated glass, fibronectin- or laminin-coated coverslips. In contrast, significantly enhanced RET between seprase and uPAR was observed on cells attached to vitronectin (Table I
). Thus, the imaging results of Figure 1
are supported by both quantification of the spectral changes (Figures 3 and 4
) and intensities (Table I
).
Single cell excitation spectrophotometry
RET between seprase and uPAR was also confirmed by single cell excitation spectrophotometry, which provides spectroscopic information complementary to those provided in the emission studies described above. In these experiments, anti-seprase mAbs were conjugated to Alexa-546, as it is better suited for excitation spectroscopy than rhodamine. Figure 5A
shows representative excitation spectra of FITC and Alexa-546. During RET the acceptors excitation spectrum gains spectral features of the donors excitation spectrum. Figure 5B
shows excitation spectra of cells adherent to fibronectin and labeled with FITCanti-uPAR and Alexa-546anti-seprase or Alexa-546-labeled anti-seprase only. RET between FITC-conjugated anti-uPAR and Alexa-546-conjugated anti-seprase on LOX cells is indicated by the appearance of two additional peaks (
485 and
505 nm) in the excitation spectrum of Alexa-546-conjugated anti-seprase. These excitation peaks are due to FITCs contribution to the RET-mediated emission of Alexa-546. Thus, several lines of evidence demonstrate the sepraseuPAR proximity complexes on LOX cell membranes. In addition to the spectroscopy experiments outlined above, the ability of certain compounds, such as cytoskeletal inhibitors, to block RET without affecting cell labeling (see below) indicate that the RET observed is not due to some photophysical or labeling artifact, but rather due to the molecular proximity of the labels.

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Fig. 5. Representative excitation spectroscopy demonstrating energy transfer from FITC-labeled uPAR to Alexa-546-labeled seprase. (A) Excitation spectra of FITC (dotted line) and Alexa-546 (dashed line) are shown. (B) LOX cells attached to fibronectin labeled with anti-uPAR and anti-seprase yield RET between chromophores (dotted line). Cells labeled with anti-seprase mAb yield Alexa-546 excitation spectrum (dashed line). The difference spectrum was obtained as a mathematical subtraction of Alexa-546 spectrum from RET spectrum (solid line). FITC and Alexa-546 peaks are labeled with arrows.
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Factors affecting the molecular proximity of seprase and uPAR molecules
Distinct membrane proteins can form domains within cell membranes due to their physical interactions with other membrane molecules, tethering to the cytoskeleton, or lipid partitioning behavior. We therefore tested the ability of several reagents to affect sepraseuPAR RET. LOX cells were treated with 104 M colchicine or 10 µM cytochalasin B followed by RET experiments, as described above. Both of these agents, which promote the disassembly of microtubules and microfilaments, respectively, abolish RET between seprase and uPAR (Figure 6
and Table I
). Thus, the cytoskeleton plays an important role in promoting molecular proximity between these two membrane molecules.

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Fig. 6. Effect of colchicine and cytochalasin B, on the RET between seprase and uPAR of LOX cells cultured on fibronectin. LOX were pretreated with each regent for 10 min and cultured for 2 h on fibronectin-coated coverslips. After fixation cells were labeled with FITC-conjugated anti-uPAR mAb and TRITC-conjugated D28 anti-seprase mAb. RET between uPAR and seprase was observed by fluorescence microscopy. Columns 14: DIC, fluorescence of anti-seprase, fluorescence of anti-uPAR, and RET. Rows 13: cells treated with 104 M colchicine, 10 µM cytochalasin B or anti-ß1 integrin (C27) mAb, respectively. Immunofluorescence and RET data were collected from three independent trials for each substrate. During each trial 3040 cells were examined (x600).
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Recent studies demonstrated association of seprase and
3ß1 integrin at invadopodia of LOX cells in a collagen-dependent manner (1) as well as ECM-dependent uPAR to ß1 integrin association at the adhesion sites of HT1080 cells (10). Therefore, we tested the effect of inhibitory anti-ß1 integrin C27 mAb on RET between seprase and uPAR. Incubation of LOX cells on the fibronectin-coated coverslips at the presence of inhibitory anti-ß1 integrin mAb resulted in the inhibition of the RET between seprase and uPAR (Figure 6
). Based on these results we conclude that sepraseuPAR supramolecular complex formation is integrin-dependent.
Detection of the gelatinolytic activity associated with LOX cell membranes
The formation of sepraseuPAR supramolecular complexes at LOX cell surfaces provides a potent means of pericellular proteolysis due to the broad substrate specificity of these complexes. Previous studies have shown that the uPA systems proteolytic activity is linked with uPAR localization on the cell membrane (26). Therefore, we sought to assess gelatinolytic activity with respect to the membrane distribution of seprase. To accomplish this, LOX cells were incubated on matrices that included DQ-gelatin, collagen III and fibronectin for 3 h. Seprase localization on LOX membranes was shown by labeling with anti-seprase TRITC-conjugated D28 mAb. Gelatinase activity cleaves DQ gelatin (fluorescein-conjugated gelatin) yielding highly fluorescent peptides (27). Using this DQ matrix, we demonstrated that gelatinolytic activity is associated with seprase regions of LOX adherence membranes (Figure 7
). Incubation of LOX cells on the DQ matrices for 3 h resulted in the proteolysis of gelatin by 46% of all examined cells. However, it is known that other proteinases posses gelatinolytic activities. Thus, uPA can degrade gelatin (28) as well as gelatinase A/MMP-2 and gelatinase B/MMP-9. Both of these MMPs were shown to be associated with adhesion membranes of LOX cells (13). Therefore, to exclude gelatinolytic effects of uPA and MMPs, LOX cells were incubated on DQ matrices including the uPA inhibitor PAI-1 or PAI-1 and the general metalloproteinase inhibitor 1,10-phenanthroline, which has no inhibitory effect on seprase activity (17). When PAI-1 is included in these matrices, the disruption of bodipy-BSA is substantially reduced (data not shown, see ref. 20), thus indicating the functional ability of this reagent. The presence of PAI-1 in the DQ matrix results in the formation of the digested matrix patterns that resemble the patterns of seprase distribution. Addition of PAI-1 and 1,10-phenanthroline gives patterns of digested matrix that overlap with the distribution of seprase (Figure 7H and I
). Quantitative analyses of intensity levels indicate that these two reagents decrease pericellular fluorescence from 35%. These findings, coupled with prior results, suggest that multiple proteolytic activities are associated with sepraseuPAR complexes.

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Fig. 7. Gelatinolytic activity of the LOX membranes in the absence and presence of the PAI-1 and 1,10-phenenthroline protease inhibitors. LOX cells attached to the DQ-gelatin matrix with and without inhibitors were examined by immunofluorescence microscopy. Cells were labeled with TRITC-conjugated D28 anti-seprase mAb (B, E and H). Degraded by gelatinases DQ-gelatin yields fluorescent products that are detected with the FITC fluorescence filter set (C, F and I). Columns 13: DIC (A, D and G), fluorescence of anti-seprase (B, E and H), fluorescence of cleaved DQ-gelatin (C, F and I). Rows 13: DQ-matrix with no inhibitors, DQ-matrix with PAI-1, and DQ-matrix with PAI-1 and o-phenanthroline. Data were collected from three independent trials for each substrate. During each trial 50 cells were examined.
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Discussion
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Tumor cell migration, invasion and metastasis to distant sites rely on the degradation of the ECM. One mechanism promoting ECM degradation is the accumulation of proteolytic enzymes at the leading edges of tumor cells. However, the ECM is composed of multiple components that can require specific enzymatic activities for their degradation. Thus, the efficiency of directed proteolysis may be dependent on the presence of enzymes with different substrate specificities (24). Two examples of such enzymes are seprase and urokinase-type plasminogen activator system, which have been associated with tumor cell invasion. Both of these membrane enzymes localize to the leading edges of tumor cells. Seprase, a surface protease, exhibits gelatinolytic activity by degrading gelatin (17). The urokinase-type plasminogen activator system has broad substrate specificity. In addition to its gelatinolytic activity, it is able to degrade ECM components such as fibronectin, laminin, vitronectin and the protein core of proteoglycans. However, the localization of various proteolytic enzymes at the leading edge is not enough. If multiple cuts are randomly made in a three-dimensional network, the network remains more or less intact with bits hanging off, but cell invasion remains difficult for purely mechanical reasons. However, if a broad spectrum of proteolytic activities are localized to a small region (
macromolecular dimensions), then many different ECM components could be simultaneously cut at the same point in space, resistance to cell extension is thereby greatly reduced. Thus, we tested the hypothesis that seprase and uPAR form supramolecular complexes in cell membranes.
Although uPAR and seprase co-localize on LOX cell plasma membranes, this does not address the molecular proximity of these molecules since the resolution of the microscope is
200 nm. Alternatively, RET provides an elegant means of detecting molecular proximity of molecules as RET is only obtained when the donor and acceptor chromophores are separated by
7 nm or less (25). When two appropriate chromophores are separated by
7 nm and properly oriented relative to one another, energy is lost by the donor and gained by the acceptor. However, RET is dependent upon the inverse sixth power of distance and thereby can only be observed at very short (
macromolecular) distances. As the magnitude of orientation factor is uncertain, we use RET as an indicator of molecular proximity, not as a spectroscopic ruler. As false positives are not possible, RET is quite sufficient for determining the nature of macromolecular proximity relationships in the membranes of tumor cells. Using multiple independent means of detecting RET, we have established the molecular proximity of seprase and uPAR in LOX cell membranes. We have shown RET using microscopic imaging, emission spectrophotometry and excitation spectrophotometry. Thus, we have performed the following: (i) Our studies have imaged the acceptors emission while exciting the donor. (ii) Emission spectrophotometry has observed the quenching of the donor in the presence of the acceptor and the enhancement of the acceptors emission in the presence of the excited donor. (iii) Excitation spectrophotometry has detected the appearance of the donors absorption characteristics in the excitation spectrum of the acceptor. Hence, under certain conditions seprase and uPAR can collect into protease-rich domains and form supramolecular complexes within the tumor cells plasma membrane.
The formation of sepraseuPAR complexes was observed on LOX cells adherent to glass and the ECM components fibronectin, laminin and vitronectin. Moreover, RET emission of sepraseuPAR complexes overlaps the seprase-rich membrane regions on LOX cells adherent to fibronectin and laminin. Quantitative RET data indicate that there is no significant difference in RET intensity between seprase and uPAR on LOX cells adherent to glass, fibronectin and laminin. However, when LOX cells are adherent to vitronectin, an increase in RET of
80% is observed. We speculate that this effect may be due to uPARs ability to bind vitronectin (29). Three possibilities can be envisioned to account for this observation. First, vitronectin enhances the number of uPAR-seprase complexes. Secondly, vitronectin leads to tighter uPARseprase complexes (thus reducing the distance between the molecules and increasing RET). Thirdly, vitronectin causes a conformational change in the uPARseprase complex that promotes better orientational overlap of the donor and acceptor molecular orbitals. Whatever the physical mechanism underlying these changes may be, it is safe to say that vitronectin, perhaps via ligation of uPAR, changes physical properties of the uPARseprase complex.
Several biological mechanisms could account for the proximity of uPAR and seprase in cell membranes. For example, independent membrane components could be drawn together by the cytoskeleton, lateral associations of the proteins in cell membranes and sequestration by lipid domains. We found that disruption of the cytoskeleton by treatment with either colchicine or cytochalasin B eliminated RET between seprase and uPAR. In addition, seprase to uPAR association is dependent on ß1 integrins. An inhibitory anti-ß1 integrin mAb eliminates RET between these proteases. This suggests that there are multiple effects promoting the formation of supramolecular complexes in cell membranes. Based on the findings presented above, we suggest that formation of sepraseuPAR complexes is dependent upon the cytoskeleton and ß1 integrins. These findings are consistent with the idea that integrins can act as supramolecular organizational centers in the plasma membrane (30).
The present studies have shown that seprase and uPAR form supramolecular structures in the membrane of LOX tumor cells. These interactions can be enhanced by vitronectin and blocked by cytoskeletal-disrupting drugs and ß1 integrin blocking. The gelatinolytic activity of seprase has been confirmed by visualizing the coincident locations of seprase and its functional activity. Although it is not technically possible to simultaneously measure uPA and seprase activity, we infer, based upon their molecular proximity, that both activities are present in these domains. Potential cross-regulation of seprase and uPAR is suggested by our studies. Moreover, it may be possible to design molecules affecting these interactions that may also affect the invasive and metastatic abilities of melanoma cells.
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Notes
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3 To whom requests for reprints should be addressed Email: hpetty{at}biology.biosci.wayne.edu 
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Acknowledgments
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This work was supported by NIH Grants: CA74120 (H.R.P.) and HL33711 (W.T.C.).
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Received March 19, 2002;