Antibodies and DNA constructs
Antibodies against the following antigens were used: EEA1, Rab5A, Rab7, Rab11, caveolin-1 (all from Santa Cruz Biotechnology, Inc.), Rab21 (Opdam et al., 2000
), β1-integrin (P5D2, P4G11, and AIIB2), α5-integrin (BIIG2), EGFR (151-IgG; all from the Drosophila Studies Hybridoma Bank), α2 (mAb MCA2025; Serotec), pAb AB1934 (Chemicon), α1 (MAB1973; Chemicon), α6 (MAB699; Chemicon), β1 (HUTS-21 [BD Biosciences] and MAB2252 [Chemicon]), collagen type 1 (RAHC11; Imtek), GFP polyclonal antibody, fluorescently conjugated secondary antibodies, Cell Tracker dyes, and labeled transferrin (all from Invitrogen).
Full-length Rab21 was subcloned from Rab21 murine cDNA (clone 6490069; IMAGE) by PCR amplification and ligated into pRluc-C2 (PerkinElmer) and in pEGFP-C2 (CLONTECH Laboratories, Inc.). Rab21GTP (Q76L), Rab21GDP (T31N), and CCSS (residues 218 and 219) mutants were generated using QuikChange Site-directed mutagenesis kit (Stratagene). Rab21 COOH-terminal–deletion mutant was generated by introducing a stop codon after E144. Plasmids encoding GFP-Rab5a, GFP-Rab7, YFP-Rab9, and GFP-Rab11 have been described (Wilcke et al., 2000
; Barbero et al., 2002
; Lebrand et al., 2002
; Gomes et al., 2003
), and YFP-mouse talin was provided by D. Critchley (University of Leicester, Leicester, UK). α2-Integrin was subcloned from the α2 cDNA in pawneo2 vector (Ivaska et al., 1999
) into pEGFP-C2 vector. The signal sequence of α2-integrin (annealed synthetic oligos corresponding to nucleotides 43–129 in the published sequence [Takada and Hemler, 1989
] was inserted to the NheI site in the vector to enable correct targeting to the plasma membrane. The GFP–α2-integrin cytoplasmic tail mutants were generated by using QuikChange Site-directed mutagenesis kit. All clones were verified by sequencing.
Rluc-tagged Rab21 constructs alone or with GFP-α2 variants (CHO cells that lack endogenous collagen binding integrins) were transfected into 95% confluent cells using Lipofectamine 2000 and incubated for 18 h. For immunoprecipitations with endogenous proteins, confluent MDA-MB-231 cells (20 × 106
) were collected from plastic plates with cold PBS. For analysis of association with cell surface–labeled integrin, MDA-MB-231 cells were plated on collagen-coated dishes for 1 h and surface biotinylated with cleavable biotin (0.5 mg/ml EZ-sulfo-NHS-SS-biotin in HANKS buffer) for 30 min on ice. After washings, cells were either lysed immediately or warmed for 15 min in HANKS +37°C to allow internalization. Cells were lysed in IP buffer (PBS with 1% octylglycoside, 0.5% BSA, 1mM CaCl2
, 1mM MgCl2
, and protease inhibitor cocktail [Roche]) on ice for 15 min. Postcentrifugation supernatant was precleared with BSA-blocked (IP buffer) protein G–agarose beads and divided into five aliquots for immunoprecipitations with different anti-Rab antibodies or a control antibody and protein G beads (90-min rotation at 4°C). After three washings (IP buffer containing 0.3% octylglycoside), SDS sample buffer was added and the proteins were separated by SDS-PAGE (4%/10%) and immunoblotted for β1-integrin (MAB2252). Reimmunoprecipitations were performed as described earlier (Mattila et al., 2005
). For the luminescent immunoprecipitations, the beads were transferred into white microtiter plate wells (96-well) and treated with Rluc substrate (5 μg/ml coelenterazine [Nanolight Technologies]), and the luminescence was measured with a multilabel HTS counter (Victor2
Integrin internalization and recycling assay
These were performed as described previously (Roberts et al., 2001
; Ivaska et al., 2002
) with some modifications. After 1 h of adhesion to collagen-coated dishes, the transfected cells were placed on ice, washed once with cold PBS, and surface labeled with 0.5 mg/ml cleavable NHS-SS-biotin (Pierce Chemical Co.). After washings, prewarmed (+37°C) HANKS medium was added, and protein traffic (internalization and recycling) was allowed to occur for the times indicated. Biotin was removed from cell surface proteins by MesNa reduction and iodoacetamide quenching on ice. The cells were lysed (200 mM NaCl, 75 mM Tris, 15 mM NaF, 1.5 mM Na3
, 7.5 mM EDTA, 7.5 mM EGTA, 1.5% Triton-X-100, and Complete), and the amount of biotinylated integrin was assayed using the anti–β1-integrin antibody AIIB2 to capture the integrins and HRP anti-biotin antibody for ELISA detection. As control, the cells were lysed after the labeling to determine the amount of total biotinylated integrin.
Stable MDA-MB-231–expressing GFP-Rab21 cells were fixed in 1% PFA with or without 0.01% glutaraldehyde in 100 mM phosphate buffer, pH 7.0, for 2 h at RT. Next, cells were pelleted in 10% gelatine and postfixed in 1% PFA for another 24 h. Ultrathin cruosections were prepared on a cryochamber (EM FCS; Leica), and thawed sections were incubated with a polyclonal antiserum raised against EGFP followed by incubation with protein A complexed to 5-nm gold particles according to standard procedures. Sections were observed in an electron microscope (model 1010; JEOL) operating at 80 kV.
Sucrose gradient fractionations
HeLa cells were transiently transfected with GFP, GFP-Rab21, or GFP-Rab21GTP using Lipofectamine 2000 as described in the Immunoprecipitations section. 48 h after transfection, the cells were harvested and fractionated on a sucrose density gradient and analyzed by Western blotting as described previously (Hughes et al., 2002
Yeast two-hybrid screen and yeast mating tests
The α2-integrin COOH-terminal tail (28 residues) Gal4 DNA binding domain fusion (pGBKT7 vector) was used to screen a mouse E17 Matchmaker cDNA library (CLONTECH Laboratories, Inc.) as described previously (Mattila et al., 2005
). In yeast mating tests, pGADT7-Rab21 (95–222) prey was transformed in Y187 host strain and cytoplasmic tails of α2- and α11-integrin (pGBKT7-α2 and -α11) and their variants in AH109 host strain. Point mutants were generated with the QuikChange Site-directed mutagenesis kit and confirmed by sequencing. The negative and positive controls in yeast mating tests were pGBKT7-53/pGADT7-T and pGBKT7/pGADT7, respectively.
Cell lines and RNAi transfections
MDA-MB-231 cells (American Type Culture Collection) were grown in DME + 1% nonessential amino acids and 10% FBS. Saos-2, HeLa, HT1080, and HEK293T cells (American Type Culture Collection) were grown in DME + 10% FBS, and PC3 cells (American Type Culture Collection) were grown in F12 medium + 10% FBS. CHO cells (American Type Culture Collection) were grown in MEM Alpha Medium + 5% FBS. Saos-2 cells express no endogenous α2 (Ivaska et al., 1999
). Stable Saos-2 cells expressing equal levels of chimeric integrins (extracellular domain of α2 fused with α1 or α5 cytoplasmic tails; Ivaska et al., 1999
) have been described (Mattila et al., 2005
). Two different annealed siRNAs targeting Rab21 (sense, ggcaucauucuuaacaaagtt and ggucaagagagauuccaugtt; Ambion) or scramble control siRNA (Silencer Negative control #1 siRNA; Ambion) were transfected at a 100-nM concentration to MDA-MB-231 or PC3 cells using Oligofectamine (Invitrogen) according to the manufacturer's protocol (48-h culture). pSilencer 4.1-CMV hygro vector (Ambion) was used to express shRNAs. Annealed DNA oligos (Scr sense strand, gatcccgcgaatcctacaagcgcgcttgatatccggcgcgctttgtaggattcgttttttccaaa; Rab21 sense strand, gatccggtcaagagagagettccatgttcaagagacatggaatctctcttgacctga) were ligated to the vector between BamHI and HindIII sites. Plasmids were verified by sequencing. shRNA plasmids and transfected into MDA-MB-231 cells using Lipofectamine 2000 (Invitrogen), and stable cell clones were generated with hygromycin selection.
Adhesion and migration assays
96-well plates were coated with collagen or fibronectin (0.25 μg/ml) overnight and blocked with 0.1% BSA (1 h, 37°C). Transiently transfected cells (GFP-Rabs or siRNA) were harvested, trypsin inhibited with 0.2% (wt/vol) Soybean trypsin inhibitor, and stained (only siRNA-transfected cells) with CellTracker Green CMFDA (Invitrogen) according to the manufacturer's instructions. Cells were suspended in 0.5% BSA in serum-free DME, seeded (5,000 cells/well) on the plates, and allowed to adhere for 30 min at 37°C. After one washing with PBS, cells were fixed (4% PFA, 10 min). Adhesion was measured by counting the number of green fluorescent cells using Acumen Assay Explorer 488 nm. The total number of GFP-positive cells was assayed after adhesion to collagen for 4 h in the presence of 10% FBS. Adhesion assays with CHO cells were done by cotransfecting GFP-α2WT or GFP-α2CYTOKR1160/61AA mutant together with Rluc-Rab21 or Rluc alone. The assays were done as described earlier in this paragraph, except that the specific adhesion time on collagen was lengthened to 1 h and 45 min.
For the scratch wound assay, stable MDA-MB-231 cells expressing GFP, GFP-Rab21, GFP-Rab21GTP, or GFP-Rab21GDP were generated. The cells were seeded onto collagen-coated 96-well plates at 35,000 cells/well and allowed to adhere overnight in the presence of 10% FBS. The wound was generated by scratching with a plastic tip. Images were taken from each well immediately and after 20 h, and the wound areas were analyzed using AxioVision 4.3 software (Carl Zeiss MicroImaging, Inc.). The number of live cells (proliferation) was scored at 0 and 20 h from identical wells using WST-1 (Roche).
For the human bone adhesion assay, transfected PC3 cells were resuspended in serum-free DME, seeded 10,000 cells/well (Cambrex OsteoAssay plate [PA-1000]), and allowed to adhere for 45 min before fixation as described earlier in this section. Green fluorescent cells were counted using a widefield epifluorescence microscope (narrow GFP filter and 20× objective). The total number of transfected cells was assayed as described earlier in this section.
Cells were plated on acid-washed glass coverslips coated with 5 μg/ml collagen type I, allowed to adhere for 1 h, washed in PBS, and PFA fixed. After permeabilization (PBS/0.02% saponin/10% FBS, 15 min), cells were stained with primary antibodies (in the same buffer) for 1 h at RT. After three washings, Alexa 488–, Alexa 555–, or Alexa 647–conjugated secondary antibodies were added (in the same buffer). Slides were examined using an inverted fluorescence microscope (Carl Zeiss MicroImaging, Inc.) or a confocal laser-scanning microscope (Axioplan 2 with LSM 510; Carl Zeiss MicroImaging, Inc.) equipped with 100×/1.4 Plan-Apochromat oil-immersion objectives. Confocal images represent a single z section of ~1.0 μm. β1-Integrin and transferrin internalization were studied as described previously (Powelka et al., 2004
A multilaser microscope (IX81; Olympus) equipped with a 488-nm TIRF condensor and a 60×/1.4 Plan-Apochromat oil-immersion objective was used for TIRFM. TIRFM was combined with conventional widefield epifluorescence microscopy and time-lapse series (frame rate ~2/s) Widefield images were pseudocolored red and TIRFM images green. Transiently transfected GFP-Rab21 cells were plated on acid-washed glass-bottomed dishes (MatTek Corporation) coated with 10 μg/ml collagen type I and allowed to adhere for 1 h before microscopy. Clear medium with 2.2 g/l NaHCO3 was used for imaging in heat (37°C) and CO2 (5%) stable environment box.
The Axioplan 2 microscope equipped with Plan-Apochromat 63× (NA 1.4) objective and a camera (Orca 2; Hamamatsu Photonics) was used for widefield epifluorescence time-lapse imaging at a rate of 2 frames/s. GFP-Rab21 and its mutant variants (or Rluc-Rab21 and GFP–α2-integrin in the cotransfection studies) were transfected to MDA- MB-231 adenocarcinoma cells. Clear DME 4500 supplemented with 1% l-glutamine, 0.5% BSA, and 30 mM Hepes was used as imaging medium. Microscopy was performed in a heat-stable environment for no longer than 1 h. MetaMorph imaging software (Universal Imaging Corp.) was used in image analysis.
Fluorescence intensities for TIRFM and widefield epifluorescence microscopy were measured and analyzed with MetaMorph software. Vesicle intensities from time-lapse series were background corrected in each time point with the formula (IB − IV) × (AV/[AB − AV]), where I is integrated intensity for region area A. B stands for background and V for vesicle. Region for vesicle (AV) was created just around vesicle and region for background (AB) just around the vesicle region. Results from two groups were compared using a t test, and statistical significance was set at P < 0.05.
Online supplemental material
Fig. S1 shows the cellular localization of endogenous β1-integrin, caveolin-1, Rab21, and EEA1 in MDA-MB-231 cells expressing GFP-Rab5 or -Rab21 and internalization of β1-integrin antibody and labeled transferrin in GFP-Rab5– and GFP-Rab21–expressing cells. Fig. S2 shows that overexpression of Rab21 does not influence the traffic of labeled transferrin in cells or the adhesion of cells to matrixes other than type I collagen. Fig. S3 shows the localization of organelle markers on the sucrose gradient–fractionated GFP-Rab21–expressing HeLa cells and immunogold electron micrographs of GFP-Rab21–positive structures in MDA-MB-231 cells. Table S1 demonstrates the association of Rab21WT and its variants with α/β1-integrin heterodimers in HT1080 cells. Video 1 shows MDA-MB-231 cells expressing GFP-Rab21, adhering to collagen recorded on GFP channel. Video 2 shows a combined widefield epifluorescence and TIRFM analysis of MDA-MB-231 cells expressing GFP-Rab21, adhering to collagen. Video 3 shows a combined widefield epifluorescence and TIRFM analysis of MDA-MB-231 cells expressing GFP-Rab21GDP mutant, adhering to collagen. Video 4 shows MDA-MB-231 cells cotransfected with GFP–α2-integrin and Rluc-Rab21WT, adhering to collagen recorded on GFP channel. Video 5 shows MDA-MB-231 cells transfected with GFP–α2-integrin alone adhering to collagen recorded on GFP channel. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200509019/DC1