Cholera toxin subunit B (CTxB) labelled with Alexa 594 (C22843) or Alexa 488 (C22841) were from Molecular Probes. Unlabelled cholera toxin B subunit (#227039), anti-cholera toxin antibody (#227040) and Brefeldin A (#203729) were obtained from Calbiochem. Latrunculin A (L5163), Nocodazole (M1404) and Alexa488-transferrin were from Sigma. Monoclonal anti-beta tubulin antibody (E7) was from Developmental Studies Hybridoma Bank. Anti-gamma tubulin and anti-caveolin1 antibodies were from Santa Cruz Biotechnology. Anti-GM130 conjugated to FITC and anti-phospho-caveolin (Tyr14) were purchased from Transduction Labs. Purified biotinylated aerolysin and anti-aerolysin antibody was from Protox Biotech. Anti Rac1 antibody was from (Upstate Biotechnology, Lake Placid, NY). Anti β1 integrin antibody was obtained from Dr. A. F. Horwitz. Anti-AMF (autocrine motility factor) receptor antibody was obtained from Dr. Ivan Nabi. Anti Arf1, Arf3, Arf4 and Arf5 specific antibodies were obtained from Dr. Richard Kahn. Temperature-sensitive Vesicular Stomatitis Virus-G-GFP was obtained from Dr. Jenniffer Lipincott-schwartz. Protein kinase D1 kinase-dead mutant (PKD~KD), PKD~KD K618N and PKD~KD P155/287G mutant constructs were obtained from Dr. Vivek Malhotra. WTRab11–GFP, (S25N) Rab11, WT Rab22a, dominant negative (S19N) Rab22a, WT cav1, Y14F cav1, WT ARF6, dominant negative (T27N) Arf6, fast cycling (T157A) Arf6 constructs and the monoclonal anti-Arf6 antibody were as described 38, 55, 56 3, 30
. RNA interference sequence 5'-AGCTGCACCGCATTATCAA-3' of rat ARF6 mRNA (GenBank accession number NM_024152) used to generate complementary small interfering RNA (siRNA) oligonucleotides annealed and inserted into pSUPER.gfp/neo were as described 57
Mouse embryonic fibroblasts from Cav1−/− and Cav1+/+ littermate mice (provided by Dr. Richard Anderson, University of Texas Health Sciences Center, Dallas TX) were cultured in DMEM medium with 10% fetal bovine serum, penicillin, and streptomycin (Invitrogen, Carlsbad, CA). Cells (106) were transfected with 40µgm of DNA (5µgm DNA plasmid + 35µgm of salmon sperm DNA) by electroporation using the GenePulser Xcell (Bio-Rad Laboratories, Hercules, CA). Cells were incubated for 6h with 5mM sodium butyrate to promote protein expression. If required, cells were serum starved 12 h after electroporation in medium with 0.2% fetal bovine serum. Cells analyzed ~24 h after electroporation generally showed 90–95% transfection efficiency. 60µg of Arf6 or scrambled dsRNA in pSUPER vector were electroporated into cells, cells incubated for 72h and then serum starved as above.
For experiments, cells were detached with 1x trypsin, treated with soybean trypsin inhibitor, washed and held in suspension with 1% methylcellulose before replating on fibronectin-coated coverslips. For aerolysin labeling experiments cells were detached with Accutase (Innovative Cell Technologies Inc.) and processed as above. Fibronectin was used at 10µg/ml except for time course experiments where it was reduced to 2µg/ml to facilitate analysis of rates of exocytosis and spreading, as is indicated in figure legends.
Analysis of surface CTxB and aerolysin
Suspended or adherent cells were placed on ice for 15min and then incubated with 10µg/ml unlabelled CTxB (Calbiochem) or 1µg/ml of biotinylated aerolysin (Protox Biotech) for 30min. Cells were washed with cold PBS and 2×105 cells were lysed in 100µl of SDS sample buffer. Cell-equivalent amounts of lysate were resolved by SDS PAGE, transferred to nitrocellulose and blocked with 5% nonfat dry milk in TBS+0.5% Tween-20 (TBS-T). Blots were incubated with anti-CTxB antibody (1µ/ml) or anti-aerolysin antibody (0.5µl/ml) followed by anti-goat or anti-mouse antibody conjugated to horse radish peroxidase respectively and developed using the ECL plus detection system (Amersham). Blots were stripped using Re-blot Plus stripping solution (Chemicon International) and re-probed with monoclonal anti-beta tubulin antibody as a loading control. Bands were quantified by densitometry using the Image J software. CTxB band intensities were normalized to tubulin.
Localisation of CTxB
Stably adherent cells were placed on ice for 15min then incubated with 10µg/ml of CTxB-Alexa 594 or 488 as indicated in Phosphate Buffered Saline (PBS) for 15 min. Cells were detached, held in suspension, replated as above and movement of endocytosed CTxB studied. In suspended or replated cells, surface GM1 was detected by incubating cells on ice with 10µg/ml CTxB-Alexa594 in PBS for 15min before fixation in 3.5% paraformaldehyde. Labeled cells were permeabilised in PBS containing 3% BSA and 0.05% Triton X100 for 15 min and blocked with 3%BSA in PBS for 1 h. Cells were then stained with anti AMF–R antibody, followed by anti-rat Alexa 488 antibody (Molecular Probes), with the anti gamma-tubulin antibody, followed by anti-mouse Alexa 488 antibody, or directly with FITC-conjugated anti-GM130 antibody. Cells expressing HA tagged Arf6 were incubated with anti-HA antibody, followed by anti-mouse IgG-Alexa488. For double labeling of CTxB-Alexa568 with tubulin, cells were fixed with cold 80% methanol for 10min at −20°C, stained with anti-β tubulin monoclonal antibody and anti-mouse IgG Alexa 488 secondary. Cells were mounted in Fluoromount-G (Southern Biotech), observed using the Zeiss LSM 510 laser confocal microscope with a 40x or 60x objective and analysed using either the Zeiss LSM Image Browser or the Image J software (NIH).
Quantitation of intracellular CTxB localisation
Cells were surface labeled with Alexa-CTxB, suspended, fixed and mounted as detailed above. Confocal images were recorded being careful to avoid pixel saturation. Using Image J software (NIH), thresholds were set to a) map the entire cell and b) to map the compact internal pool of endocytosed CTxB. The tracing tool was then used to select edges of the thresholded areas. Total intensity within the thresholded areas was determined, and the percentage of CTxB within the internal pool and the rest of the cell calculated.
Co-labelling of cells with transferrin and CTxB
Stably adherent cells were incubated with 20µg/ml transferrin-Alexa594 (Sigma) for 30min, washed and chilled on ice for 15min. Cells were then incubated with 10µg/ml of Alexa488-CTxB (Molecular Probes) in DMEM for 30min. Cells were washed with DMEM, detached and held in suspension with 1% methylcellulose in DMEM. At the end of the incubation they were fixed or replated on FN as described above.
Quantitation of endocytosed transferrin distribution
Cells labeled with transferrin-Alexa594 as above and replated were fixed, mounted and confocal images recorded being careful to avoid pixel saturation. Using Image J software (NIH), thresholds were set to a) map the entire cell and b) to map the endocytosed transferrin and the thresholded area for each calculated. Area occupied by transferrin was calculated as percentage of total cell area.
Stably adherent cells were incubated for 30min with 1µM Latrunculin A, detached with trypsin, held in suspension for 90 min with 1% methylcellulose, fixed or replated on FN and fixed with 3.5% paraformaldehyde. For BFA treatment, cells suspended for 90min were incubated for 30min with 1µg/ml BFA in suspension and fixed immediately or replated on FN prior to fixation. For early NOC treatment, stably adherent cells were incubated for 10min with 10µM NOC, detached with trypsin, washed with DMEM, and held in suspension for 90min with 1% methylcellulose in DMEM in the continuous presence of the drug. For late NOC treatment, 10µM NOC was added to cells after 90 min in suspension. Control cells were left untreated. All cells were incubated for another 30min in suspension, washed to remove methylcellulose and replated on coverslips coated with 2µg/ml FN with or without NOC. After 15 min, cells were washed, fixed and mounted as before. Cells were observed using a Nikon Diaphot TMD fluorescence microscope using a 60X oil objective and photographed using a Coolsnap HQ camera (Roper Scientific).
ARF6 activity assay
Arf6 activity was measured as previously described 36
. Stably adherent cells were detached, held in suspension 2h and replated on 10µg/ml FN. Cells were lysed on ice in buffer containing 50 mM Tris, pH 7.5, 100 mM NaCl, 2 mM MgCl2
, 1% Triton X-100, and protease and phosphatase inhibitor cocktails (Calbiochem). Lysates were centrifuged for 30min at 13,000xg and incubated with 40µg GST-GGA3 plus 10µl glutathione-Sepharose beads (Amersham) for 30 min. at 4°C. Beads were washed thrice in lysis buffer and eluted with SDS sample buffer. Arf6 was detected by Western blotting with anti Arf6 antibody 36
Cells were washed with cold phosphate-buffered saline, scraped, and homogenized with 500µl of buffer containing 10 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 1 mM DTT, 0.25 M sucrose, and a cocktail of protease inhibitors (Sigma, St. Louis, MO). Nuclei and unbroken cells were removed by centrifugation at 1000 × g for 10 min at 4°C, and the postnuclear supernatant was centrifuged at 100,000 × g for 1 h at 4°C to sediment the membranes. 60% of membrane and 1.6% of cytosolic fractions were analyzed by Western blotting for Rac1 and β1 integrin. Rac1 levels in the membrane fraction were normalized to β1 integrin levels.
Rac1 activity assay
Rac1 pull down assays for GTP loading were performed essentially as described 35
. 60% of GST-PBD bound active Rac1 and 1.6% of whole cell lysates were analyzed by Western blotting and bands quantified by densitometry. The amount of active Rac was calculated relative to total Rac for each sample.
Measuring cell spreading
Images of adherent cells were analyzed using Image J software. Thresholds were set to map the entire cell, the tracing tool was used to select the edge of each thresholded cell. The total area within the mapped edge for each cell was determined.
Quantitation of early CTxB endocytosis in suspended cells
Cells photographed using the Laser Confocal Microscope were analysed using Image J. By thresholding and using the “percentage shrink” macro, 20% of cell area from the cell periphery (≈0.5–0.7µm) was defined as cell edge. Central 40% area of the cell was defined as cell center. Intensities in these regions were measured, normalized to area and represented as percentage of total CTxB intensity.
Comparison between data points were done using the Student’s t test (Sigmaplot Stastical Analysis Software).
Fluorescent Image Colocalization Analysis
Images of cells photographed using the Laser Confocal Microscope at both wavelengths were analysed using Image J software (NIH), and Pearson’s coefficient determined using the colocalization threshold plugin. Intensity quantitation along a defined line for each wavelength determined using the ‘plot profile’ function. List of values obtained were plotted in sigmaplot.