Cells and DNA constructs
HeLa cells and primary HFFs (gift from J. Roberts, Fred Hutchinson Cancer Research Center, Seattle, WA) were cultured in DME supplemented with 10% FBS and 1% penicillin-streptomycin. HeLa cells were transduced to stably express control or Dab2 shRNA. A pBabe puro vector with the histone H1 promoter cloned into the second long terminal repeat (Welcker et al., 2003
) was modified by swapping in the hygromycin selection gene (gift from M. Maurer, Fred Hutchinson Cancer Research Center). Hairpins to target Dab2 or a control sequence were inserted downstream of the H1 promoter 5′-CAAAGGATGTGGGTCAACATT-3′. The control sequence targeted was 5′-TATGTCAAGTTGTATAGTTA-3′.
Retroviral particles were generated by cotransfection of HEK293T cells with constructs and packaging vector. HeLa cells were infected with retrovirus and, 48 h later, selected with 250 µg/ml hygromycin. Protein expression was detected by immunoblotting. For rescue experiments, vector alone (pCGT) or vector encoding T7-tagged mouse Dab2 (p96 or p67) was cotransfected with pGL1SuperC GFP expression vector into HeLa cells using Lipofectamine 2000 (Invitrogen).
The following integrin antibodies were provided by E. Wayner (Fred Hutchinson Cancer Research Center): inhibitory mouse anti–integrin β1 (P5D2-1), mouse anti–integrin α5 (P1D6-H9), and mouse anti–integrin αvβ5 (P1F6). Other antibodies included mouse anti–integrin α1 (TS2/7; Santa Cruz Biotechnology, Inc.), rabbit anti-Dab2 (Santa Cruz Biotechnology, Inc.), mouse anti-Dab2 (BD), mouse anticlathrin (X22; Abcam), mouse antiadaptin (clone AP.6; EMD), mouse anti-T7 (EMD), mouse anti-TfnR (Ab-1; Abcam), rabbit anti–phospho FAK (pY576; Invitrogen), rabbit anti-FAK (Santa Cruz Biotechnology, Inc.), rabbit anti–phospho Src (pY416; Cell Signaling Technology), rabbit anti-Numb (C29G11; Cell Signaling Technology), rabbit anti-ARH (gift from L. Traub, University of Pittsburgh, Pittsburgh, PA), rabbit anti-Fyn (FYN3; Santa Cruz Biotechnology, Inc.), mouse antivinculin (hVIN-1; Sigma-Aldrich), mouse anti-EEA1 (BD), rabbit anti-EEA1 (Thermo Fisher Scientific), and mouse anti–extracellular signal-regulated kinase (BD). The mouse hybridoma cell line (LP-016) expressing monoclonal Src antibody (LA074) was a gift from J. Meisenhelder (Salk Institute for Biological Studies, La Jolla, CA), and the 327 anti-Src mouse monoclonal antibody was provided by J. Brugge (Harvard Medical School, Boston, MA). Alexa Fluor–tagged secondary antibodies were purchased from Invitrogen.
The CSC method for suspension cells was previously described (Wollscheid et al., 2009
). The following modifications were made for adherent cells and quantitative analysis (Ong et al., 2002
). Control and Dab2-deficient HeLa cell lines were grown in normal and heavy lysine/arginine-supplemented media (Invitrogen), respectively, for five to six population doublings. When just confluent, cells were removed from 10 15-cm plates using 2 mM EDTA in PBS for 10 min at 37°C, harvested by centrifugation, and allowed to recover in suspension in the respective media at 37°C for 30 min. Equal cell numbers were then combined and washed once with cold PBS and once with cold labeling buffer (PBS containing 0.1% BSA and 20 mM Pipes, pH 6.7). All subsequent steps were performed at 0–4°C. Cells were oxidized with 1.2 mM NaIO4
in 40 ml of labeling buffer for 30 min, washed in cold PBS, and incubated in 25 mM biocytin hydrazide (Biotium, Inc.) in 10 ml of labeling buffer for 2 h. Cells were washed twice, resuspended in 20 ml of hypotonic buffer (10 mM Tris HCl, pH 7.5, and 0.5 mM MgCl2
) for 10 min, and broken with 20 strokes of a tight Dounce homogenizer. A postnuclear supernatant was centrifuged in the SW41 rotor (Beckman Coulter) at 35,000 rpm for 1 h. Membrane pellets were resuspended and recentrifuged to remove cytoplasmic contamination. Subsequent steps of dissolving in Rapigest, digesting with Endo Lys-C and trypsin, binding to streptavidin beads, washing at high pH, and eluting bound N
-glycosylated peptides with protein N
-glycosidase F have been described previously (Wollscheid et al., 2009
). Samples were analyzed using mass spectrometers (QTOF [Agilent Technologies]; Finnigan LTQ-FT [Thermo Fisher Scientific]). Peptides were identified by using the SEQUEST algorithm (Eng et al., 1994
) in combination with Peptide Prophet (Nesvizhskii et al., 2003
), searching for peptides containing aspartic acid instead of asparagine in NXS/T glycosylation signals. ASAP (automated statistical analysis of protein abundance) ratios were determined (Li et al., 2003
) and curated manually.
Steady-state surface and total integrin levels were measured by flow cytometry. Cells were detached using 10 mM EDTA-PBS for 10 min at 37°C, washed with PBS, pelleted by centrifugation, and fixed in cold 4% paraformaldehyde-PBS for 20 min. Cells were incubated with anti-integrin or TfnR antibodies for 1 h at room temperature. To measure total integrin, cells were permeabilized with 0.1% Triton X-100 in PBS for 5 min at 25°C before incubating with anti-integrin antibodies. Surface or total antibody was quantified by FACS analysis after staining with an Alexa Fluor 488 goat anti–mouse secondary antibody. Profiles were gated on intact cells, based on morphology, and mean fluorescent intensity was obtained. For rescue experiments, profiles were gated on GFP-positive cells, and surface antibody was quantified after staining with an Alexa Fluor 647 goat anti–mouse secondary antibody.
Knockdown experiments in HeLa cells and primary HFFs were performed as described previously (Maurer and Cooper, 2006
). Cells were transfected with 50 pmol of a pool of four siRNA oligonucleotides specific for Dab2, clathrin, AP2 µ2, or ARH (Thermo Fisher Scientific) using Oligofectamine (Invitrogen) on days 1 and 3 and analyzed on day 5. Numb siRNA (Thermo Fisher Scientific) was given on days 1 and 3, and cells were analyzed on day 6. Total cell lysates were analyzed by immunoblotting to show that target protein levels were significantly reduced compared with a protein loading control ().
For rescue experiments, siRNA specific for human Dab2 (Thermo Fisher Scientific) was used to deplete cells of Dab2. Cells were transfected with T7-tagged mouse Dab2 on day 4 and analyzed on day 5. For partial knockdown of integrin β1, cells were transfected with 20 pmol of siRNA to integrin β1 (Santa Cruz Biotechnology, Inc.) on day 1 and analyzed on day 2.
Integrin β1 RNA was measured as described previously (Laszlo and Cooper, 2009
). Total RNA was extracted from 106
cells using TRIZOL reagent (Invitrogen). Total RNA was reverse transcribed using SuperScript II reverse transcription and random primers (Invitrogen). PCR was performed using Taq polymerase and the following primers: 5′-CCCTTGCACAAGTGAACAGA-3′ and 5′-ACATTCCTCCAGCCAATCAG-3′ (integrin β1) or 5′-GCGAGAAGATGACCCAGATCATGTT-3′ and 5′-GCTTCTCCTTAATGTCACGCACGAT-3′ (actin).
Antibody internalization assay
Antibody uptake was measured using a modification of a previous method (Roberts et al., 2001
). Cells were plated on coverslips coated with 4 µg/ml collagen IV and then incubated with anti–integrin β1 antibody (P5D2) and/or Alexa Fluor 488–conjugated human Tfn diluted in assay media (DME, 10 mM Hepes, pH 7.4, and 0.1% BSA) for 30 min at 4°C. After washing off unbound antibody/Tfn with cold DME, cells were warmed to 37°C in DME with 10% FBS for 0, 15, or 30 min and fixed with cold 4% paraformaldehyde-PBS for 20 min. To visualize internalized receptors, surface-bound antibody/Tfn was removed by acid stripping for 5 min on ice (0.5 M NaCl and 0.2 M acetic acid) before fixing with cold 4% paraformaldehyde-PBS.
Integrin recycling was measured as described previously (Roberts et al., 2001
). Cells were washed twice with cold PBS before incubation with 0.2 mg/ml sulpho-NHS-SS-biotin (Thermo Fisher Scientific) in PBS for 30 min at 4°C. After surface labeling, cells were washed with cold PBS and transferred to DME with 10% FBS for 0, 15, or 30 min at 37°C to allow internalization. At the indicated times, cells were washed twice with cold PBS and then treated with 20 mM MesNa (50 mM Tris, pH 8.6, and 100 mM NaCl) for 15 min at 4°C to remove biotin. MesNa was quenched with 20 mM Iodoacetamide (50 mM Tris, pH 8.6, and 100 mM NaCl) for 10 min at 4°C. After two cold PBS washes, cells were lysed (75 mM Tris, pH 7.4, 200 mM NaCl, 15 mM NaF, 7.5 mM EDTA, 1.5% Triton X-100, 1 µg/ml aprotinin, 5 µg/ml leupeptin). Lysates were clarified by centrifugation at 15,000 g
for 15 min. Supernatants were incubated with anti–integrin β1 antibody (P5D2), and immunoprecipitates were analyzed by SDS-PAGE.
To visualize surface integrin, nonpermeabilized cells were fixed and stained with anti-integrin antibodies to extracellular epitopes. To detect intracellular proteins, cells were fixed and permeabilized with 0.1% Triton X-100 in PBS for 5 min at 25°C. Cells were blocked in a solution of 2% BSA, 5% normal goat serum, and PBS for 30 min at 25°C. Primary antibodies were diluted in blocking solution and added either for 3–4 h at 25°C or overnight at 4°C. Coverslips were rinsed three times in PBS before the addition of Alexa Fluor 488–, Alexa Fluor 568–, or Alexa Fluor 647–labeled secondary antibodies, diluted 1:1,000 (all from Invitrogen), for 1 h at 25°C. Secondary antibodies included goat anti–mouse, goat anti–mouse IgG1-isotype–specific and IgG2b-isotype–specific antibodies, and goat anti–rabbit. After several PBS rinses, coverslips were mounted in ProLong Gold solution (Invitrogen).
Cells were visualized using a 60× NA 1.42 oil objective on a microscope (DeltaVision IX70; Olympus). Images were recorded using fixed camera settings using a microscope (IX-HLSH100; Olympus). Images were acquired and deconvolved using SoftWoRx software (Applied Precision, LLC). Deconvolved images from single planes corresponding to the ventral surfaces of the cells or flattened z projections were analyzed using ImageJ (National Institutes of Health). Figures were assembled using Photoshop (Adobe) and Canvas (Deneba) softwares. Levels were adjusted equally for all images in a set.
Collagen uptake assay
Cells were detached using 10 mM EDTA-PBS for 10 min at 37°C, washed with PBS, pelleted by centrifugation, and plated on coverslips coated with 4 µg/ml Alexa Fluor 488 collagen IV at 37°C in DME containing 10% FBS. Cells were fixed with 4% paraformaldehyde-PBS after 2 h, permeabilized with 0.1% Triton X-100, and stained.
Boyden chamber assay.
Migration assays were performed in a 48-well micro chemotaxis chamber (Neuro Probe, Inc). Cells were detached using 10 mM EDTA-PBS for 10 min at 37°C, washed three times with DME, and resuspended in DME at 250,000 cells/ml. The lower wells of the chamber were loaded with 10% FBS in DME. An 8-µm pore diameter membrane (Neuro Probe, Inc.) coated with 4 µg/ml collagen IV or 2 µg/ml vitronectin separated the bottom and top chambers. Cells were added to the top wells. The chamber was incubated in a humidified atmosphere of 5% CO2 at 37°C for 12 h. The cells on the top side of the membrane were removed, and the migrated cells on the bottom side were stained with 0.1% crystal violet in 20% ethanol and counted by using a light microscope at a magnification of 40.
Scratch wound assay.
Dab2 shRNA and control cells were grown at equivalent density for 24 h on coverslips coated with 4 µg/ml collagen IV. A single scratch was made with a pipette tip, and cells were allowed to migrate into the wound for 10 h. Cells were stained with antibodies to integrin β1 and vinculin and visualized using a 40× oil objective on a microscope (DeltaVision; Carl Zeiss, Inc.).
Online supplemental material
Fig. S1 shows that steady-state surface levels of integrin β1 increase upon depletion of Dab2. Fig. S2 shows that Dab2 regulates integrin α1β1 endocytosis and collagen uptake. Fig. S3 shows that Dab2 and integrin colocalize over the entire dorsal surface, whereas Numb is enriched at the periphery of the cell on the ventral side. Fig. S4 shows that the combined removal of Dab2 and Numb caused a greater inhibition of migration than removing either alone. Fig. S5 shows that FAK and Src activation increases on Dab2 depletion. Table S1 shows isotope labeling ratios (ASAP; light/heavy) of 35 cell surface–labeled proteins, excluding integrins, that were quantified by SILAC and CSC. Table S2 shows surface integrin quantification by SILAC labeling and CSC of proteins from control cells labeled with light isotopes and Dab2-deficient cells labeled with heavy isotopes. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200812160/DC1