Src-dependent phosphorylation of caveolin-1 Tyr-14 promotes swelling and release of caveolae.

Cell cultures

Early passage (4–8) HLMVECs from Lonza (Walkerville, MD) were seeded on 0.2% gelatin-coated dishes in endothelial basal medium 2 (EBM2) supplemented with EGM-2-MV BulletKit (Lonza), 10% FBS, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and penicillin/streptomycin. RLMVECs (passages 12–20) from Vec Technologies (Rensselaer, NY) and HEK 293 cells (American Type Culture Collection, Manassas, VA) were grown in DMEM (GIBCO, Lombard, IL) supplemented with 10% FBS, 10 mM HEPES, penicillin/streptomycin, and, as required to maintain stable expression, G418 (GIBCO). Isolation of Cav1 − / − mouse lung endothelial cells Cav1 − / − mice obtained from Jackson Labs (Farmington, CT) were used to isolate lung endothelial cells according to an approved Institutional Animal Care and Use protocol. Neonatal mouse lungs (3 d postnatal) were used to generate highly enriched MLEC cultures by magnetic bead separation with anti-CD31 Ab as described ( Tiruppathi et al ., 2000 ; Sverdlov et al ., 2009 ). The isolated endothelial cells, positive for acetylated low-density lipoprotein, platelet endothelial cell adhesion molecule 1, and angiotensin-converting enzyme, were grown on 0.1% gelatin-coated tissue culture plates or glass coverslips in EGM-2 containing 15% FBS and 20 mg/ml endothelial cell growth supplement and used at passage 2.

Reagents

All reagents were obtained from Sigma-Aldrich (Milwaukee, WI) unless stated otherwise. PP2 and N -nitro- l -arginine methyl ester hydrochloride were obtained from Calbiochem (San Diego, CA), and ODG was purchased from RPI (Mt. Prospect, IL). Caveolin-1 pAb and pY14-caveolin-1 mAb were from BD Biosciences (San Jose, CA), β-actin and normal mouse immunoglobulin G (IgG mouse) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and pY118-paxillin was obtained from Thermo Scientific (Waltham, MA). 4,6-Diamidino-2-phenylindole (DAPI), Alexa 488– and Alexa568–labeled BSA, and all fluorescently labeled secondary antibodies were purchased from Invitrogen (Waltham, MA). Horseradish peroxidase–conjugated goat anti-mouse and goat anti-rabbit secondary antibodies were from Kirkegaard and Perry Laboratories (Gaithersburg, MD).

Cloning of caveolin-1 constructs

Human caveolin-1 WT cDNA in pcDNA 3.1 ( Minshall et al ., 2000 ) was subcloned into pcDNA 6 containing Myc and histidine tags and used as a template to generate mutants Y14F, Y14D, and C156S using a two-step PCR protocol as described ( Andersson et al ., 1998 ). Primer pairs were designed and used to introduce site-specific mutations in codons 14 and 156 (see the following list). To generate the Y14D mutation, two separate reactions with primer pairs 1/4 and 2/3 were used to introduce site-specific mutations in codon 14 while amplifying either the region 5′ or 3′ of the base pair change. The resulting PCR products were gel purified and used as overlapping templates in the second-step reaction with primer pair 1/2 to generate the full-length Y14D mutant. The Y14F and C156S constructs were generated similarly using primer pairs 1/6 and 2/5 for the Y14F mutation and 1/8 and 2/7 for the C156S mutation. All constructs were sequenced to confirm presence of mutations. We used the following primers pairs: Forward WT-Cav, 5′-CTAGGTACCCAGCATGTCTGGGGGC­AAAT-3′ Reverse WT-Cav, 5′-CCTAGGCCTCGAGTATTTCTTTCTGCAA­GTTG-3′ Forward Y14D, 5′-GGACATCTCGACACCGTTCCCAT-3′ Reverse Y14D, 5′-ATGGGAACGGTGTCGAGATGTCC-3′ Forward Y14F, 5′-GGACATCTCTTTACCGTTCCCAT-3′ Reverse Y14F, 5′-ATGGGAACGGTAAAGAGATCTCC-3′ Forward C156S, 5′-CACACCGTCTCTGACCCACTC-3′ Reverse C156S, 5′-GAGTGGGTCAGAGACGGTGTG-3′ To generate C-terminal–tagged EGFP, EYFP, and ECFP Cav1 constructs for 4D TIRF and for FRET experiments (referred to, respectively, as Cav1-GFP, Cav1-YFP, and Cav1-CFP), we subcloned Cav1 into pEYFP-N1 or pEGFP-N1 and pAmCyan vectors (Clontech, Mountain View, CA) via restriction sites 5′- Nhe I and 3′- Kpn I and sequenced them. To obtain YPET and CyPET-tagged WT-Cav1 constructs, we amplified YPET and CyPET (AddGene, Cambridge, MA) from the original vectors by PCR with addition of Bam HI and Not I restriction sites. PCR products were then digested and subcloned into WT-Cav1 pEYFP-N1 vector previously digested with Bam HI and Not I enzymes, so that the EYFP tag was excised and substituted with either CyPET or YPET. Substitution of tags was verified by sequencing. Cell transfections Transfection of cDNA was performed by electroporation using an Amaxa Nucleofection kit (Amaxa, Gaithersburg, MD) or by Lipofectamine 2000 reagent (Invitrogen), according to manufacturer’s instructions. Cells were used for experiments 24–48 h after transfection.

Cell treatments

Cells were washed with 37°C Dulbecco’s phosphate buffered solution (DPBS) buffer supplemented with calcium and magnesium, followed by starvation in serum-free growth medium for 3 h. As indicated, cells were pretreated with 25 μM Na 3 VO 4 or 10 μM PP2 for 15 min before stimulation with BSA (3 mg/ml) for 20 min. Cells were placed on ice and washed once with ice-cold phosphate-buffered saline (PBS), three times with acid wash buffer (0.2 M acetic acid and 0.5 M NaCl, pH 2.5), and once with PBS before performing cell lysis. Western blot analysis For Western blotting, samples were lysed on ice in the presence of protease inhibitors (P8340; Sigma-Aldrich) and phosphatase inhibitor cocktails 2 and 3 (P5726 and P0044; Sigma-Aldrich). The less stringent sample preparation conditions included mechanical disruption of cells by passage through a 21-gauge needle in HB buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA) supplemented with 2% ODG. The unbroken cells and nuclei were removed by low spin (500 × g for 10 min), and supernatants were taken for determination of protein concentration by the BCA Protein Assay Kit (Pierce, Rockford, IL). Samples for Western blotting were prepared in NuPage sample buffer with or without dithiothreitol (DTT; nonreducing conditions) and heated at 60°C for 10 min before loading. The more stringent (“standard”) sample preparation conditions included solubilization in 1× RIPA buffer (BP-115; Boston BioProducts, Ashland, MA), followed by disruption with sonication (Sonic Dismembrator 100; Fisher Scientific, Hanover Park, IL). Under stringent conditions, samples were made in 1× Laemmli sample buffer (#161-0747; Bio-Rad, Hercules, CA) supplemented with 10 mM DTT and boiled (100°C for 5 min) before loading onto the gels. Proteins were then separated on either 12% SDS–PAGE gel or NuPage gradient gel using appropriate running and transfer buffers (Novex, Waltham, MA).

Immunoprecipitation

Cells from at least one confluent 100-mm dish were collected and solubilized in HB buffer supplemented with 2% ODG plus protease and phosphatase inhibitors as described. All insoluble materials were removed by centrifugation at 10,000 rpm (5415R; Eppendorf) for 10 min. The soluble supernatant was collected, and a 10% aliquot was saved for input analysis by Western blot. The rest of supernatant was incubated with 5 μg of primary anti-Myc antibody (ab9106; Abcam) O/N at 4°C with end-over-end rotation, followed by incubation with protein A/G agarose beads (Santa Cruz Biotechnology) for 1 h with rotation at 4°C. Beads were spun down and washed three times with ice-cold ODG lysis buffer, once with high-salt buffer (0.5 M NaCl), and once with PBS. Proteins were eluted from the beads by incubation for 20 min with 1× Laemmli sample buffer supplemented with DTT, followed by boiling at 100°C for 5 min. Samples were run on SDS–PAGE gels for Western blot analysis. Fractionation by density gradient centrifugation Fractionation was conducted as described previously ( Macdonald and Pike, 2005 ) with modifications. In brief, two confluent 10-cm plates were washed with PBS and scraped into 0.6 ml of ice-cold homogenization buffer (25 mM MES [2-( N -morpholino)ethanesulfonic acid], pH 6.5, 150 mM NaCl, 60 mM n -octylglucoside with protease inhibitors). Cells were broken using a Dounce homogenizer, followed by passage through a 21-gauge needle. Homogenates were spun briefly at 500 × g to remove unbroken cells, and the cleared supernatant was loaded on top of 4.2 ml of a 5–30% sucrose gradient prepared in the same lysis buffer and centrifuged in SW55Ti rotor (Beckman, Indianapolis, IN) for 16 h at 50,000 rpm. Eleven equal fractions were collected from the top of the gradient, and samples were separated by SDS–PAGE gels, followed by Western blotting with anti–caveolin-1 Ab or anti–phosphoY14-caveolin-1 Ab.

Isolation of cytosolic and membrane fractions

To separate soluble and insoluble fractions, cells were grown to confluence on 10-cm plates, starved, and treated as indicated. Cells were then placed on ice, washed twice with PBS and/or acid wash buffer (if BSA treated), scraped, pelleted at 500 × g for 10 min, and stored at –20°C. Cell pellets were thawed and suspended in ice-cold, detergent-free buffer (10 mM Tris-HCl, pH 7.4, 2 mM EDTA, 250 mM sucrose) supplemented with protease and phosphatase inhibitor cocktails. Cells were broken by Dounce homogenizer or by passing through a 27-gauge needle. Unbroken cells and nuclei were removed by centrifugation at 500 × g for 10 min. The clarified supernatant was spun in a TLA-55 rotor (Beckman) for 30 min at 50,000 rpm. The pellets corresponding to the membrane fraction (insoluble proteins) and the supernatant (cytosolic proteins and microvesicles) were adjusted to the same concentration in HB buffer supplemented with 2% ODG and then analyzed by Western blotting. Antennapedia-conjugated CSD peptide A peptide corresponding to the CSD (amino acids 82–101) was synthesized (bioWORLD, Dublin, OH) as a biotin-labeled conjugate together with the Antp internalization sequence as described ( Bucci et al ., 2000 ). Characterization of the dose and time dependence of peptide internalization in cultured RLMVEC indicated that maximal uptake was achieved by 1 h of incubation at 37°C with 5 μM peptide. Control experiments used the biotin-labeled Antp peptide without the CSD. RapR-Src activation RapR-Src and mCherry FRB retroviral constructs were generated as described ( Chu et al ., 2014 ).

HLMVEC cells

(Lonza) were infected with RapR-Src and FRB viral supernatants for 24 h, after which the cells were starved and stimulated with 0.5 μM rapamycin to activate Src for 0–60 min. 125 I-albumin uptake and transendothelial transport Confluent cells were serum starved for 3 h and then incubated at 37°C with 0.1 mg/ml of tracer-labeled 125 I-albumin (PerkinElmer-Cetus, Waltham, MA) in the presence or absence of 100 mg/ml of unlabeled albumin to measure total versus nonspecific uptake as previously described ( John et al ., 2003 ). Uptake assays were stopped by placing cells on ice and adding ice-cold Hanks balanced salt solution (HBSS) followed by two additional washes with ice-cold HBSS and two washes with acid wash buffer (0.2 M acetic acid and 0.5 M NaCl, pH 2.5) to remove cell surface–bound 125 I-albumin. Cells were then lysed, resuspended, and transferred to vials for γ counting to determine the amount of radioactivity (counts per minute [cpm]) inside the cells. For measurement of transendothelial (vesicle-mediated) permeability of 125 I-albumin across RLMVEC monolayers, cells were grown on porous Transwell filter inserts (12-mm diameter, 1-cm 2 growth area, 0.4-μm pore size; Corning Costar, Cambridge, MA) until fully confluent. Where indicated, cells were washed with PBS and loaded for 1 h at 37°C with 5 μM Antp-CSD peptide or Antp alone, followed by extensive washes with PBS. Cells were then starved for 3 h in serum-free growth medium. The transcytosis assay was performed by adding 0.1 mg/ml albumin labeled with 1 × 10 5 cpm 125 I-albumin in the presence or absence of unlabeled 100 mg/ml albumin added to the upper chamber of Transwells. The amount of 125 I-albumin transported from the top to the bottom chamber across the RLMVEC monolayer was then measured as described previously ( John et al ., 2003 ). Alexa 488–BSA uptake Confluent Cav1 − / − MLECs were washed with PBS, incubated for 3 h in serum-free medium, and incubated with Alexa 488–conjugated albumin (0.1 mg/ml Alexa 488–BSA [Invitrogen] mixed into 1 mg/ml of nonfluorescent BSA in HEPES-buffered HBSS solution for 30 min at 37°C. Subsequently the cells were washed extensively with ice-cold PBS, followed by acid wash buffer (0.2 M acetic acid and 0.5 M NaCl, pH 2.5) to remove noninternalized/membrane-associated albumin. The cells were then fixed, permeabilized, and stained with anti-Myc or anti-caveolin pAb and the nuclear marker DAPI ( Shajahan et al ., 2004a ). Nonconfocal DAPI images were acquired using Hg-lamp excitation and an ultraviolet filter set; confocal microscopy was performed using a Zeiss LSM 510 META system with a 100×/1.45 numerical aperture (NA) AlphaFluor objective and 488/543-nm excitation laser lines. Fluorescence emission was detected in optical sections of