Heparanase facilitates cell adhesion and spreading by clustering of cell surface heparan sulfate proteoglycans.

Antibodies and reagents Anti syndecan-1 monoclonal antibody

(B-B4) was purchased from Serotec (Oxford, UK). Antibodies to syndecan-4 and paxillin were purchased from Santa Cruz Biotechnology (Sanata Cruz, CA). Anti-Rac 1 monoclonal antibody was purchased from Becton Dickinson Biosciences (San Diego, CA), and PAK PBD-agarose beads were from Cell Biolabs Inc (San Diego, CA). Anti-vinculin monoclonal antibody, phalloidin-TRITC, heparin, fibronectin, and the 110 kDa fibronectin-like genetically engineered protein, were purchased from Sigma (St. Louis, MO). Avidin-FITC was purchased from Vector (Burlingame, CA). The selective p38 (SB-203580), Src (PP2), Erk (PD-98059), PKC [Bisindolylmaleimide I (Bis)], Rock (Y27632) and Rac (NSC 23766) inhibitors were purchased from Calbiochem (San Diego, CA) and were dissolved in DMSO as stock solutions. DMSO was added to the cell culture as a control. The KKDC and its control, scrambled peptide were synthesized as described previously [29] . The introduced C-terminal cysteine residue mediates spontaneous peptide dimerization that can be further facilitated by enhanced oxygenation generated by stirring. For biotin labeling, an extra lysine residue was introduced at position 12 to create the sequence KKFKNSTYSRS K biotin SVDC. This approach was undertaken in order to preserve the N-terminal lysine residues critical for the peptide interaction with HS [29] . A peptide from the COOH-terminal heparin-binding domain of fibronectin (Hep II peptide, WQPPRARI) [38] was purchased from Sigma (St. Louis, MI). Cells and cell culture U87 MG human glioma and Colo 320 colon carcinoma cells were purchased from the American Type Culture Collection (ATCC). Rat C6 glioma cells were kindly provided by Dr. Eli Keshet (The Hebrew University Hadassah Medical School, Jerusalem) [39] , and human skin fibroblasts were kindly provided by Dr. H. Mendel (Rambam Health Care Campus, Haifa). ARH-77 leukemia-derived F cells were kindly provided by Dr. Ben-Zion Katz (Soraski Medical Center, Tel-Aviv, Israel). F cells are a sub-population of the ARH-77 cell line which poorly adhere and mainly floats (F) once plated on fibronectin-coated dishes [40] . Cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with glutamine, pyruvate, antibiotics and 10% fetal calf serum in a humidified atmosphere containing 5% CO 2 at 37°C. Wild type Chinese hamster ovary (CHO) K1 and mutant cells (pgs A-745) deficient in xylosyltransferase and unable to initiate glycosaminoglycan synthesis, were kindly provided by Dr. Jeffery Esko (University of California, San Diego), and grown in RPMI 1640 medium supplemented with 10% FCS and antibiotics [39] . Recombinant wild type and enzymatically-inactive heparanase mutated at glutamic acid residues 225 and 343 [double mutant (DM)] critical for enzymatic catalysis [41] were purified from the conditioned medium of HEK 293 transfected cells, essentially as described [42] .

Cell morphology and immunocytochemistry

Cells were plated on glass cover slips uncoated or coated with fibronectin or the 110 kDa fibronectin-like protein for the time indicated in the absence (Con) or presence of the indicated peptides [scrambled (Scr), or KKDC; (50 µg/ml)], proteins [latent 65 kDa or mutated, enzymatically inactive heparanase (1 µg/ml)], or inhibitors. Cells were then fixed with 4% paraformaldehyde in PBS for 15 minutes, and visualized. For immunofluorescent staining, cells were fixed with cold methanol for 10 minutes, washed with PBS and subsequently incubated in PBS containing 10% normal goat serum for 1 hour at room temperature, followed by 2 hours incubation with the indicated primary antibody. Cells were then extensively washed with PBS and incubated with the relevant Cy2/Cy3-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA) for 1 hour, washed and mounted (Vectashield, Vector, Burlingame, CA). Staining was observed under a fluorescent confocal microscope. For actin staining, cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% triton X-100 for 2 minutes, washed and incubated with TRITC-phalloidin (Sigma) for 30 minutes and visualized by confocal microscopy, as described [43] , [44] .

Cell lysates and protein blotting

Cell cultures were incubated for 24 hours under serum-free conditions, pretreated with 1 mM orthovanadate for 10 minutes at 37°C, washed twice with ice cold PBS containing 1 mM orthovanadate and scraped into lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton-X100, 1 mM orththovanadate, 1 mM PMSF) containing a cocktail of protease inhibitors (Roche, Mannheim, Germany). Total cellular protein concentration was determined by the BCA assay, according to the manufacturer's (Pierce, Rockford, IL) instructions. Thirty micrograms of cellular protein were subjected to SDS polyacrylamid gel (SDS-PAGE) and immunoblotting, as described [29] , [45] . For estimation of Rac1 activation, lysis buffer included 50 mM Tris-HCl, pH 7.4, 300 mM NaCl, 10 mM MgCl, 4% glycerol, and 1% Triton X-100. The amount of GTP-bound Rac1 was analyzed by incubating total cell lysates (200 µg) with 20 µg of the p21-binding domain (PBD) of PAK-agarose beads. Following 30 minutes incubation, the beads were washed and, after electrophoresis and blotting, membranes were probed with anti-Rac1 antibodies [26] .

Time Lapse microscopy

Cells were grown on Corning/Nunc 6 wells dishes in DMEM medium and viewed with inverted motorized microscope (DMIRE2 Leica, Germany). Images were captured using cooled B/W CCD camera (Retiga EXI, Qimaging, USA). Time-lapse acquisition was made with custom written software (Provided by Uri Alon, Weizmann Institute, Rehovot, Israel), inserted into Image-Pro software (MediaCybernetics, USA). The system is equipped with an on stage incubator (Cube & Box, LIS, Switzerland).

Supporting Information Movie S1 Time lapse microscopy. Rat C6 glioma cells (2×104) were plated in a 6-well plate in complete growth medium, followed by 20 hours incubation in serum-free medium. Cells were then incubated with control scrambled peptide. Six fields in each well were randomly selected, and were examined every 10 min for 10 hours by the time lapse system. Representative time lapse movie is shown. (6.15 MB AVI) Click here for additional data file. Movie S2 Time lapse microscopy. Rat C6 glioma cells (2×104) were plated in a 6-well plate in complete growth medium, followed by 20 hours incubation in serum-free medium. Cells were then incubated with heparanase (1 µg/ml); Six fields in each well were randomly selected, and were examined every 10 min for 10 hours by the time lapse system. Representative time lapse movie is shown. (7.14 MB AVI) Click here for additional data file. Movie S3 Time lapse microscopy. Rat C6 glioma cells (2×104) were plated in a 6-well plate in complete growth medium, followed by 20 hours incubation in serum-free medium. Cells were then incubated with the KKDC peptide; Six fields in each well were randomly selected, and were examined every 10 min for 10 hours by the time lapse system. Representative time lapse movie is shown. (6.99 MB AVI) Click here for additional data file. Movie S4 Time lapse microscopy. Rat C6 glioma cells (2×104) were plated in a 6-well plate in complete growth medium, followed by 20 hours incubation in serum-free medium. Cells were then incubated with the KKDC peptide with heparin (10µg/ml); Six fields in each well were randomly selected, and were examined every 10 min for 10 hours by the time lapse system. Representative time lapse movie is shown. (6.15 MB AVI) Click here for additional data file. Figure S1 KKDC peptide dimerization significantly improves heparin binding and anti-heparanase properties. A. Heparin binding. KKDC peptide was synthesized in the absence (KKDC) or presence of ethandithiol (EDT), which binds covalently to the sulfate group of cysteine and prevents di-sulfide bridge formation between cysteine residues. Peptides (50 µM) were incubated (2 hours, 4°C) with heparin-Sepharose beads in PBS, washed with PBS supplemented with NaCl to a final concentration of 0.35 M, followed by one wash with PBS. Dye-free sample buffer was added and the beads were boiled for 5 minuntes, centrifuged and the supernatants were loaded on Tris-Tricine gel. Subsequently, gels were stained with Coomassie blue to visualize bound peptides (arrow). B. Heparanase enzymatic activity. B16 melanoma cells (2×106) were resuspended in RPMI medium and incubated (18 hours, 37°C) with 35S-labeled ECM in the absence (filled rectangle) or presence of control scrambled peptide dimer (green rectangle; 50 µM), KKDC peptide undergoing spontaneous dimerization (filled triangle; 50 µM), KKDC peptide following enhanced dimerization (blue rectangle; 50 µM), or heparin (filled circle; 15 µg/ml). The incubation medium (1 ml) containing sulfate labeled degradation fragments was subjected to gel filtration on a Sepharose CL-6B column. Fractions (0.2 ml) were eluted with PBS and their radioactivity counted in a beta-scintillation counter. Degradation fragments of HS side chains are eluted at 0.5