Cell Culture and Antibodies
Eph4 cells were obtained from H. Beug (Research Institute of Molecular Pathology, Vienna, Austria) and were grown in DME supplemented with 2 mM glutamine, 10% heat-inactivated FCS, 100 U/ml penicillin, and 10 μg/ml streptomycin. For growth on polycarbonate filters, 0.4 μm pore diameter, 12 mm filter diameter (Transwell; Costar Corp., Cambridge, MA), cells were trypsinized, a single cell suspension was made, and seeded at a density of 2.5 × 105 per filter. Medium was placed on both surfaces, 0.5 ml apically and 1.5 ml basolaterally, and was changed daily.
BHK-21 cells were cultured in Glasgow MEM supplemented with 5% heat-inactivated FCS, 10% tryptose phosphate, 2 mM glutamine, 100 U/ml penicillin, and 10 μg/ml streptomycin. All media and reagents for cell culture were purchased from GIBCO BRL (Eggenheim, Germany).
Zo-1 antibody R26.4C was obtained from Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA). The antibody for the FcLR 5-27 chimeric receptor was purified from 2.4G2 hybridoma supernatant (Unkeless, 1979
) by ammonium sulfate precipitation. Briefly, the 2.4G2 hybridoma supernatant was adjusted to 55% saturation with (NH4
(Sigma Chemical Co.
, St. Louis, MO). The precipitate collected by centrifugation was dissolved in H2
O (20 ml of H2
O per liter of supernatant) and dialyzed against protein G binding buffer (10 mM Na2HPO4
buffer, pH 7.0, 10 mM EDTA, 150 mM NaCl). A preswollen gamma bind plus sepharose column (Pharmacia
Diagnostics AB, Uppsala, Sweden) was equilibrated with binding buffer, the sample was applied and bound antibodies were eluted with 0.5 M acetic acid/NH3
, pH 2.5. 1 ml fractions were collected into tubes containing the appropriate volume of 2 M Tris base.
An antibody was raised against a synthetic peptide corresponding to rab17 NH2
-terminal amino acids NH2
-MAQAAGLPQASTASQPK-COOH. The 49-1 antibody was raised against the entire rab17 protein. Rab17 wild type (WT) was cloned into pRsetA vector (Invitrogen, Carlsbad, CA), transformed into Bl21 DE3 cells, and the expressed protein was purified by nickel agarose affinity chromatography and used to immunize rabbits. For immunofluorescence, the serum was affinity purified using nitrocellulose strips of the recombinant protein. For Western blot analysis the crude serum was used. The antibody against cellubrevin was kindly provided by P. De Camilli (Yale University, New Haven, CT; Galli et al., 1994
). All secondary antibodies were obtained from Dianova (Hamburg, Germany).
Preparation and Biotinylation of the Fab Fragment
The purified 2.4G2 IgG was dialyzed against papain buffer (0.1 M NaAcetate, pH 5.5). 6 mg of 2.4G2 IgG were digested with insoluble enzyme as described by the manufacturer (Sigma Chemical Co). Fab fragments were biotinylated with NHS-LC-biotin or NHS-SS-biotin (Pierce, Rockford, IL) after the protocol described by the manufacturer (ImmunoPure NHS-LC-biotinylation Kit; Pierce).
Colocalization of Rab17 and Internalized FITC-Labeled Transferrin in Nonpolarized Cells
DNAs encoding rab17 WT and the human transferrin receptor (Zerial et al., 1986
) were cloned into pGEM-1 vector (Promega Corp.
, Madison, WI) under the T7 promoter. BHK cells were trypsinized 24 h before transfection and seeded onto 11 mm coverslips. Cell were infected with T7 RNA polymerase recombinant vaccinia virus (Stenmark et al., 1995
) and cotransfected with both plasmids using DOTAP (Boehringer Mannheim
, Mannheim, Germany). 4 h after transfection of the cells in medium devoid of serum the coverslips were incubated in a humidified chamber with 50 μg/ml FITC-labeled transferrin (Ullrich et al., 1993
) for 15, 30, or 60 min at 37°C. Coverslips were then rinsed in PBS and fixed in 3% paraformaldehyde, permeabilized with 0.1% Triton X-100, and indirect immunofluorescence was performed to detect rab17. Samples were visualized by confocal microscopy.
Generation of MX-Rab 17 and MX-FcLDL5-27 Expression Constructs
The Mx promoter was excised as a 1.7-kb fragment EcoRI-BamHI from pMXhGH (Hug et al., 1988
) and inserted into p163/7 (Woodroofe et al., 1992
), from which the H2 promoter had been excised. Mx-rab17 constructs were generated by PCR. A consensus Kozak site (Kozak, 1987
) was introduced 5′ to the ATG, and NheI sites were introduced to clone the open reading frame into the Mx expression cassette. The mutations in the coding region of rab17 to generate rab17Q77L and rab17N132I were carried out by PCR-based mutagenesis according to the method of Landt et al. (1990)
using oligos 5′-GGGACACAGCCTTCCT
GGAGAAGTAC-3′ (rab17Q77L) and 5′-GCTGGTCGGCAT
The FcLR 5-27 chimeric receptor (Matter et al., 1993
)–coding region fragment EcoRI-XbaI was excised after changing by PCR the consensus Kozak site 5′ to the upstream methionine, using oligos Fc-KOZ (5′-GAT CGAATTCGCCGCCGCCATGGGAATCCTGCCGTTC-3′) and Fc-Xho primer (5′-CGGGGGCTCGAGTTTGACCACA-3′), and inserted in EcoRI-XbaI sites of pGEM-1 vector (Promega Corp.
, Madison, WI). Subsequently, the pGEM1 construct was digested with EcoRI-HincII and ligated into the Mx expression cassette EcoRI-SnaBI.
Generation and Characterization of Stable Inducible Eph4 Cell Lines
Eph4 cells were cotransfected in suspension with constructs Mx-rab17 WT, Mx-rab17N132I, or Mx-rab17Q77L and SV40neo for selection using DOTAP (Boehringer Mannheim). Colonies were selected and maintained in 800 μg/ml G418 (geneticin; GIBCO BRL). Expressing colonies were screened by indirect immunofluorescence after induction with interferon (Sigma Chemical Co.). Positive clones were expanded, plated on filter supports (Costar Corp.), and transepithelial resistance was measured. Only those lines with a transepithelial resistance above 2,000 ohm/cm2 were used for subsequent studies. Zo-1 was also visualized to ensure that the lines were polarized. The lines were subcloned to obtain a high number of expressing cells.
A second round of transfection was carried out to introduce Mx-FcLR 5-27 into the above cell lines. Cotransfection was carried out as above using cells in suspension and SV40-puro (Clontech Laboratories Inc., Palo Alto, CA) as a selectable marker. The colonies were selected in 800 μg/ml G418 and 1 μg/ml puromycin (Sigma Chemical Co.). Clones were screened by indirect immunofluorescence for both rab17 (antibody 49-1) and FcLR 5-27 (monoclonal 2.4G2 antibody). The above polarity checks were then carried out. Clones were expanded and frozen; all assays were performed on early passage cells. Levels of rab17 before and after induction with interferon were also analyzed by Western blot analysis.
Eph4 cells were seeded onto six-well plastic dishes and grown to 80% confluence. One well was induced with interferon and the other left uninduced as a control. After the induction period, the cells were washed in PBS and lysed in 200 μl PBS, 1% SDS, 0.3 mM PMSF (Sigma Chemical Co.), sonicated, boiled for 10 min, and resuspended in loading buffer. Protein content was assayed using the BCA protein assay reagents (Pierce) and BSA as a standard. 20 μg total protein was loaded onto a 12% SDS-PAGE, blotted, and hybridized with a rabbit polyclonal antibody (49-1).
Localization of Rab17 WT Protein in Polarized Eph4 Cells
Eph4 cells, harboring Mx-rab17 WT construct, were grown on filter supports; rab17 was induced with interferon and filters were rinsed in PBS+ (PBS with 1 mM CaCl2 and 1 mM Mg Cl2) at 37°C, fixed in 3% paraformaldehyde for 15 min at room temperature (RT), permeabilized in 0.1% Triton X-100 for 4 min, and quenched in 50 mM ammonium chloride for 15 min. Filters were then cut from the supports, blocked in 10% FCS for 1 h at RT in a humidified chamber, and rab17 was visualized using an affinity-purified polyclonal rabbit antibody (49-1) followed by FITC-labeled donkey anti–rabbit IgG. Nuclei were visualized with propidium iodide at a concentration of 0.1 μg/ml (Sigma Chemical Co.) for 15 min at RT, after 30 min at RT in 1 mg/ml DNase free- RNaseA (Sigma Chemical Co.). Filter pieces were mounted in 50% glycerol, 100 mg/ml DABCO (Sigma Chemical Co.) with spacers, and sealed with nail varnish. Samples were then analyzed by confocal microscopy.
Rab17 localization was also assayed by immunoelectron microscopy on plastic grown Eph4 cell line harboring Mx-rab17 WT as previously described (Chavrier et al., 1990
Colocalization of Rab17 and Internalized FITC-labeled Transferrin in Filter-grown Eph4 Cells
Eph4 cells harboring Mx-rab17 WT were grown on filter supports, and expression of rab17 was induced by the addition of interferon for 12 h. The coding region of the human transferrin receptor (hTR) was cloned into the amplicon vector pHSV (Geller and Breakefield, 1988
). pHSV- hTR was then packaged into HSV particles as previously described (Lim et al., 1996
) and used to infect Mx-rab17 WT-transfected Eph4 cells grown on filter supports (Murphy et al., 1997
). Cells were infected in serum-free medium (to deplete transferrin) for 4 h, washed briefly at 37°C, and 50 μg/ml FITC-labeled transferrin was added to the basolateral surface for 90 min at 37°C in a humidified chamber. Cells were then washed briefly, fixed in 3% paraformaldehyde, and rab17 was visualized by indirect immunofluorescence as described below. In the case of the experiment shown in Fig. , Eph4 cells were grown on plastic, and human holo-Tf (Sigma Chemical Co.
) was internalized for the indicated times at 37°C. After fixation, Tf was visualized using a sheep anti–human Tf serum followed by FITC- labeled anti–sheep IgG and Rab17 with a polyclonal rabbit antibody followed by rhodamine-labeled donkey anti–rabbit secondary antibody.
Figure 5 Colocalization of rab17 WT and internalized holo-transferrin in Eph4 cells grown on plastic. Holo Tf was internalized for 5 (A), 15 (B), 30 (C), and 60 min (D) at 37°C in Eph4 cells harboring Mx-rab17 WT after induction with interferon. Inset (more ...)
Colocalization of Rab17 and Internalized Fab Fragment in Eph4 Cells
Fab fragment was prepared as outlined above (preparation and biotinylation of the Fab fragment). Eph4 cells harboring Mx-rab17 WT were plated on coverslips and treated with interferon to induce the expression of both rab17 and the FcLR 5-27. Fab fragment was added to the cells in a volume of 200 μl at a concentration of 7 μg/ml for 5, 10, 20, 40, and 60 min at 37°C. Cells were then rinsed in PBS, fixed, and processed for indirect immunofluorescence. The Fab fragment was visualized using an affinity-purified goat anti–rat IgG F(ab′)2 fragment specific antibody followed by rhodamine-labeled donkey anti–goat secondary antibody. Rab17 was visualized using a polyclonal rabbit antibody followed by FITC-labeled donkey anti–rabbit IgG. Samples were analyzed by confocal microscopy.
Confocal Microscopy, Data Acquisition, and Analysis
Samples were visualized using the EMBL confocal scanning laser beam microscope. Confocal series were carried out at either 0.5 or 1 μm steps. The 529 and 476 laser lines of the Argon-ion laser were used for excitation of rhodamine/propidium iodide and fluorescein, respectively. In the case of Fig. , the data were collected on a Leica confocal TCS-NT microscope. Images were recorded and imported into either Adobe Photoshop or NIH Image graphic programs for compilation, and printed directly using the Kodak Color Ease printer.
Endocytosis, Transcytosis, and Recycling of the FcLR 5-27 Chimeric Receptor
Endocytosis and transcytosis mediated by chimeric receptors were assayed as previously described (Hunziker and Mellman, 1989
). Briefly, Fab fragments derived from monoclonal antibody 2.4G2 (Unkeless, 1979
) were biotinylated with NHS-LC-biotin (Pierce
, Rockford, IL) as described above (preparation and biotinylation of Fab fragment section).
Saturating concentrations of biotinylated-Fab fragments (0.5–10 μg/ml) were prebound apically or basolaterally to filter grown Eph4 cells for 1 h on ice. Unbound antibody fragments were removed by washing five times with PBS+/0.5% BSA and twice with PBS+. To determine the apical– basolateral surface distribution of the FcLR 5-27 chimeric receptor, the cell surface–associated Fab fragments were eluted with medium containing 0.5% BSA, pH 3.4. To measure intracellular transport, after binding, the cells were incubated for different lengths of time in MEM without phenol red (Sigma Chemical Co.) containing 10 mM Hepes, pH 7.4, and subsequently cooled down on ice. Apically and basolaterally bound antibodies were eluted with medium containing 0.5% BSA, pH 3.4. After 10 min, eluates were collected and elution was repeated once. The pH of these eluates was adjusted to 7.0 by adding 40–60 μl of 1 M Hepes, pH 7.6. The intracellular signal associated with the filters was determined by lysing the cells in KOAc Buffer (115 mM KOAc, 25 mM Hepes, pH 7.4, 2.5 mM MgCl2) containing 1% Triton X-100 and 0.5% SDS. Electrochemiluminescence (ECL) counts of the eluates and lysates were determined as described below. Uptake values represent the amount of signal resistant to acid elution from both surfaces. Transcytosis is calculated as the percentage of signal in the corresponding acid eluate relative to the total amount of originally bound Fab fragment.
To measure recycling of the chimeric receptor to the apical plasma membrane, saturating concentration of Fab fragments reversibly derivatized with biotin (see above) were first preinternalized apically for 10 min at 37°C. The medium was then removed and the cells were further incubated for 10 min at 37°C in the presence of 100-fold excess of nonbiotinylated Fab. Cells were then cooled on ice, washed five times with PBS+
/0.5% BSA, and biotin bound to Fab fragments remaining at the cells surface was stripped by reduction with MesNa (three washes in freshly prepared 50 mM MesNa, 100 mM NaCl, 2.5 mM CaCl2
, 50 mM Tris-HCl, pH 7.8, for 20 min followed by three washes with PBS+
containing 20 mM iodoacetamide; Matter et al., 1993
). The cells were incubated in complete medium for various times at 37°C. Biotinylated-Fab fragments, recycled to the apical membrane or transcytozed to the basolateral surface, were eluted and the ECL counts of the eluates and of the lysates were determined as described below.
Nocodazole (Sigma Chemical Co.
) was dissolved in DMSO at 33 mM stock solution in DMSO, stored at −20°C. Polarized Eph4 cells were either pretreated 3 h at 37°C or 60 min at 4°C after allowing the internalization from the basolateral side of biotinylated Fab fragments for 30 min at 37°C in the presence of 33 μm nocodazole. The apical and basolateral antibody fragments were eluted and quantified. In control experiments, Eph4 cells were incubated in the presence of DMSO alone. Nocodazole did not increase the paracellular diffusion of the Fab fragments in either direction, suggesting that the drug did not affect the integrity of the cell monolayer. Immunofluorescence was also performed to visualize the extent of microtubule depolymerization under the experimental conditions used. Samples were fixed with paraformaldehyde using the pH shift protocol (Bomsel et al., 1989
Analysis of Transferrin Transport
Iron-saturated human transferrin (Sigma Chemical Co.
) was biotinylated with NHS-SS-biotin (Pierce) as previously described (Smythe et al., 1992
). Eph4 cells grown either on plastic or on filter supports were serum starved 8 h in DME containing 10 mM Hepes and 0.2% BSA in the presence or absence of interferon. For single round of transferrin cycle experiments, cells were first incubated with 125 nM of biotinylated transferrin in internalization medium (DME, 10 mM Hepes, 0.5% BSA) for 1 h on ice (surface binding). The biotinylated transferrin was added to the basolateral side in the case of filter grown Eph4 cells. After washing six times with cold PBS+
/0.5% BSA cells were incubated with internalization medium for various times at 37°C. The medium of plastic-grown Eph4 cells or the apical and basolateral media of filter-grown Eph4 cells was collected and the surface-bound biotinylated transferrin was stripped by reduction with MesNa (Matter et al., 1993
). The intracellular signal associated with the filters was determined by lysing the cells as described above. For continuous uptake experiments, biotinylated transferrin was added to the basolateral medium of filter-grown Eph4 cells for 90 min at 37°C. The apical medium was collected, the cells were extensively washed, and then lysed. The ECL counts of the lysates, and collected media were determined as described below.
The leakage of the cells was controlled in each experiment both with the transferrin receptor and the FcLR 5-27 receptor. In experiments where the ligand was prebound on either side of the monolayer on ice, the excess ligand was washed away and the filters incubated at 37°C for various time intervals. We checked the initial leakage after prebinding by stripping the ligand from both cell surfaces at time zero after washing away excess ligand in duplicate filters. After prebinding and washing away excess ligand, the filters were incubated at 37°C for various lengths of time. For each time point at 37°C, duplicate filters were incubated on ice. The leakage was controlled by stripping the ligand from both cell surfaces at the same time as the 37°C samples were processed. In the experiments where the continuous uptake of transferrin for 90 min at 37°C was quantified, duplicate filters were incubated on ice to check leakage of the monolayer.
Electrochemiluminescence Detection System
The ECL-Analyser System was purchased from Igen Inc. (Rockville, MD). Affinity-purified goat anti–rat IgG F(ab′)2 fragment specific antibody and rabbit anti–sheep IgG were labeled with Origen Tag-NHS (N-hydroxysuccinimide ester of a ruthenium chelate; Igen, Inc.) following the protocol described by the manufacturer (Igen, Inc.). The ECL counts of the surface-bound (acid-releasable) and intracellular (acid-resistant) biotinylated Fab for the transcytotic assay were determined as follow: 50 μl of the eluates and lysates were incubated with 4 μg/ml ruthenium- labeled affinity-purified goat anti–rat IgG F(ab′)2 fragment specific antibody for 2 h at RT. Thereafter, 1 μl of M-280 Streptavidin Dynabeads (Dynal, Hamburg, Germany) was added for 15 min at RT. The reaction was stopped by adding 200 μl of assay buffer (Igen Inc.) and samples were directly quantified by the ECL analyzer.
With respect to transferrin trafficking, a different protocol was set up. The samples containing biotinylated transferrin were incubated first with 1 μl of M-280 streptavidin Dynabeads, then with 1.25 μl of sheep anti–human transferrin serum (SAPU, Law Hospital Carluke, Scotland) diluted in 400 μl of wash buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2% Triton X-100, 0.2% BSA), and then finally with 2 μg/ml ruthenium-labeled rabbit anti–sheep IgG. All the incubation steps were performed at RT for 1 h. Between the different incubation steps, M-280 streptavidin Dynabeads were washed three times with wash buffer using a Dynal MPC-E Magnetic Particle Concentrator (Dynal, Oslo, Norway). After the last washing step the Dynabeads were resuspended in 100 μl of wash buffer and 100 μl of assay buffer and subsequently quantified by the ECL-analyzer. Both assays were validated by comparing the results to those obtained using iodinated Fab fragment and iodinated transferrin.