Materials:
Table 1
Antibodies used in this project
Antibodies | Experiment |
rabbit anti-EFR3A (Abnova GmbH, Taipei City, Taiwan) | WB 1:1000 |
mouse anti-Flotillin 2 (Santa Cruz Biotechnology, Dallas, TX USA ) | WB 1:1000; IP: 5µg |
mouse anti-FLAG (Merck KGaA, Darmstadt, Germany) | WB 1:1000 |
rabbit anti-EGFR (Cell Signaling, Danvers, MA, USA) | WB 1:1000 |
mouse anti-Flotillin 1 (Santa Cruz Biotechnology, Dallas, TX USA) | WB 1:1000 |
goat anti-Flotillin 2 (Abnova GmbH, Taipei City, Taiwan) | WB 1:1000; IP: 5µg |
mouse anti-GAPDH (Santa Cruz Biotechnology, Dallas, TX USA ) | WB 1:1000 |
goat IgG (Santa Cruz Biotechnology, Dallas, TX, USA) | IP: 5µg |
mouse IgG (Santa Cruz Biotechnology, Dallas, Dallas, TX, USA) | IP: 5µg |
Anti-Rabbit IgG (HRP) (Jackson ImmunoResearch, Ely, Cambridgeshire, United Kingdom) | WB 1:10000 |
Anti-Mouse IgG (HRP) (Jackson ImmunoResearch, Ely, Cambridgeshire, United Kingdom) | WB 1:10000 |
Anti-Goat IgG (HRP) (Santa Cruz Biotechnology, Dallas, Dallas, TX, USA) | WB 1:10000 |
rabbit polyclonal anti-phospho PLCγ1 Y783 (Cell Signaling, Danvers, MA, USA) | WB 1:1000 |
rabbit polyclonal anti-PLCγ1 (Cell Signaling, Danvers, MA, USA) | WB 1:1000 |
rabbit monoclonal Phospho-EGF Receptor (Tyr1068) (D7A5) (Cell Signaling, Danvers, MA, USA) | WB 1:1000 |
rabbit monoclonal EGF Receptor (D38B1) (Cell Signaling, Danvers, MA, USA) | WB 1:1000 |
Reagents
A list of all antibodies used in this study can be found in Table 1. Protein G Dynabeads, di-4 ANEPPDHQ (Invitrogen, D36802), Bodipy SM, propidium iodide – 1.0 mg/ml, Pierce High Capacity Ni-IMAC Resin, EDTA compatible, Fluo-4 Direct Calcium Assay Kit, and Amplex Red Cholesterol Assay Kit were from Thermo Fisher Scientific Waltham, MA USA. DMEM (Dulbecco's Modified Eagle Medium) without L-glutamine, Fetal Bovine Serum (FBS), glutamine GlutaMAX (100X), 100 units/ml penicillin and 100 µg/ml streptomycin, 1x phosphate buffered saline (PBS), trypsin–EDTA 0.05%, and Hank's Balanced Salt Solution (HBSS) were from Gibco (Waltham, MA, USA). Puromycin was from Santa Cruz (Dallas, TX, USA). cOmplete Protease Inhibitor Cocktail was from Roche (Basel, Switzerland). Radiance ECL was from Azure Biosystems (Dublin, CA, USA). DMSO (dimethyl sulfoxide; methyl sulfoxide), ribonuclease A (RNase A) 10 mg/ml, and glycine were from VWR (Radnor, PA, USA). Nitrocellulose membranes were from Amersham, GE Healthcare Life (Chalfont St Giles, UK). Kanamycin was from Bioshop (Burlington, ON, Canada). Of the remaining reagents and materials, octyl β-D-glucopyranoside, cyanogen bromide-activated-Sepharose 4B, methyl-β-cyclodextrin (MβCD), NEM (N-ethylmaleimide), lysozyme, imidazole, 1,4-dithiolthreitol (DTT), BCA Protein Assay Kit, and Tween 20 were from Merck (Sigma) KGaA (Darmstadt, Germany), while 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acid (HEPES), isopropyl β-d-1-thiogalactopyranoside (IPTG), phenylmethylsulfonyl fluoride (PMSF), sodium dodecyl sulfate (SDS), Tris HCl, Triton X-100, sucrose, and ROTIPHORESE NF-Acrylamide/Bis-solution 30 (29:1) were from Roth (Karlsruhe, Germany).
Cell culture
The HeLa cell line used in this study was purchased from ATCC and cells were cultured in DMEM medium containing 10% fetal bovine serum, 2 mM glutamine GlutaMAX, 100 units/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO2. HeLa EFR3A-knockdown (KnD) and HeLa “scrambled” control were cultured in the same medium additionally supplemented with 2 µg/ml puromycin at 37°C in a humidified atmosphere of 5% CO2.
Detergent-resistant membrane
The DRM (detergent-resistant membrane) fraction was isolated from 25x106 HeLa cells which were washed with TNE buffer (10 mM Tris HCl, 150 mM NaCl, 5 mM EDTA, pH 7.5). Protease inhibitor cocktail was added and cells were resuspended in 300 µl of ice-cold DRM isolation buffer (10 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100), incubated on ice for 20 min, occasionally vortexed and mixed with an equal volume of 80% sucrose in the same buffer. Finally, samples were gently applied on the top of a discontinuous sucrose gradient composed of 2.7 ml of 30% sucrose and 0.9 ml of 5% sucrose and ultracentrifuged for 16 h, at 35,000 RPM, 165,000xg, 4°C, using an Optima L90K, ultracentrifuge with a SW-60 Ti rotor (Beckman Coulter, Brea, CA, USA). After ultracentrifugation 10 fractions (420 µl) were collected from the top of the gradient.
Lentiviral transduction and transient transfections
HeLa EFR3A knockdown (KnD) and HeLa scrambled were performed using EFR3A shRNA Lentiviral Particles (sc-77469-V) prepared by Santa Cruz Biotechnology (Dallas, Texas USA) according to the manufacturer’s protocol. Transient transfections of cells were performed using CLB (Lonza, Basel, Switzerland) electroporation equipment according to the manufacturer’s protocol for HeLa cells, using program 4 as recommended.
Expression of recombinant proteins
His-tagged recombinant flotillin-2 was purified under denaturation conditions as described previously [28]. Human EFR3A cDNA clone in a bacterial expression vector based on the pPB-N-His vector, containing a single N-terminal 6X-histidine tag was ordered from genomics-online.com, Aachen, Germany. EFR3A was expressed in E. coli strain NiCo21(DE3) and purified under native conditions according to the previously published protocol with slight modifications [39]. Initially, cells were precultured overnight at 37°C, shaking at 180 rpm in 6 ml of LB medium containing kanamycin (35 µg/ml). Then, 400 ml of fresh LB medium with kanamycin (35 µg/ml) was inoculated with overnight preculture and grown at 37°C to reach optical density of the culture of 0.6 at 600 nm. Recombinant protein induction was carried out using isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 0.5 mM at 18°C with constant shaking (180 rpm) for 16 h. After this time bacteria were collected by centrifugation at 10000xg/15 min. was resuspended in a lysis buffer, 10 mM HEPES, 500 mM NaCl, 1mM PMSF, cOmplete Protease Inhibitor Cocktail, 10 mM imidazole, 0.5% Triton X-100, 25 U/ml nuclease OMNI, and 1 mg/ml lysozyme. The obtained suspension was homogenized via pressing through a needle (0.9 x 40 mm) followed by sonication in a Hielscher sonicator (UP100H) 10 times for 0.5 s with 0.5 s intervals on ice, 80% amplitude. Bacterial lysate was centrifugated at 30000 x g for 30 min. The obtained supernatant was mixed with Pierce High Capacity Ni-IMAC Resin, EDTA compatible resin which was washed before 3 times with a lysis buffer. The mixture was incubated at 4°C for 3 h then placed into the column and washed 3 times with a wash buffer 1: 10 mM HEPES, 500 mM NaCl, 10 mM imidazole, pH 8.0, followed by wash buffer 2: 10 mM HEPES, 300 mM NaCl, 10 mM imidazole, pH 8.0, until the A280 dropped below 0.05. Protein was eluted with 300 mM imidazole 10 mM HEPES, 300 mM NaCl, pH 7.2. Peak fractions of the protein were analyzed by SDS–PAGE (10% gel). The gel was stained with Coomassie, or Western blot was performed.
Pull-down assay
Recombinant flotillin-2 was dialyzed against 0.1 M NaHCO3, 0.5 M NaCl pH 8.5 overnight at 4°C. Flotillin 2 was mixed at 1:1 with the above buffer containing 2% octyl β-D-glucopyranoside, pH 8.5 and next covalently conjugated to CNBr Sepharose 4B according to the manufacturer’s protocol. Control resin was prepared by incubation with 0.1 M NaHCO3, 0.5 M NaCl, 1% octyl β-D-glucopyranoside, pH 8.5. In order to quench possibly remaining active groups, flotillin-2-conjugated and control Sepharose 4B resin were incubated with 0.2 M glycine, pH 8.0 for 16 hours. Prepared resins were stored at 4°C.
Resins prepared as above (700 µl) were centrifuged and then washed 3 times with buffer (20 mM Tris pH 7.4, 150 mM NaCl, 1% octyl β-D-glucopyranoside, 1 mM EDTA, protease inhibitor cocktail, centrifuged every time at 1000 x g, for 1 min at 4°C. Both resins were incubated in 0.1% BSA in 20 mM Tris pH 7.4, 150 mM NaCl, 1% octyl β-D-glucopyranoside, 1 mM EDTA, protease inhibitor cocktail overnight at 4°C, then washed 10 times by centrifugation at 1000xg/1min with “lysis buffer” (50 mM Tris pH 7.5, 300 mM NaCl, 2% octyl β-D-glucopyranoside, protease inhibitor cocktail, 2 mM EDTA). DRM fractions were mixed at a 1:1 ratio with the lysis buffer, kept on ice for 30 min (with occasional gently vortexing) and incubated for 16 hours with flotillin-2-Sepharose or control resins, with gentle shaking on a CappRondo CRR-08X Blood mixer roller. After this time resins were washed 8 times with lysis buffer, then bound proteins were eluted with 2 × SDS sample buffer (20% SDS, 50% glycerol, 25 mM EDTA, 250 mM DTT, 0.25 M Tris pH 6.8) and separated by SDS–PAGE (10% gel). Gels were stained with Coomassie and next the gel fragment containing protein bands of molecular mass greater than 60 kDa was cut out and subjected to MS/MS identification performed by the MS laboratory at the Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
Co-immunoprecipitation
9x106 HeLa cells were solubilized in 20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 5% glycerol, 2% octyl β-D-glucopyranoside pH 7.4, protease inhibitor cocktail for 30 min on ice and incubated with 5 µg of goat anti-flotillin 2 antibodies coupled to Protein G Dynabeads at 4°C overnight, with gentle shaking on a Rotator SB2 (Stuart). Nonimmune Goat IgG (5 µg) was used as a control. The beads were washed 1x PBS-T (1x phosphate buffered saline (Gibco), 0.1% Tween 20) and eluted with 5 × SDS sample buffer (20% SDS, 50% glycerol, 25 mM EDTA, 250 mM DTT, 0.25 M Tris pH 6.8) and separated by SDS-PAGE (10% gel) and analyzed by Western blot with appropriate antibodies.
MβCD treatment of cells
HeLa cells were grown as mentioned above for 48 h to confluency, then washed with PBS and incubated for 1 h at 37°C with DMEM medium supplemented with 10% FBS, 2mM glutamine and 1x PBS in the presence (treated cells) or absence (control cells) of 8 mM MβCD. Then cells were treated with trypsin–EDTA 0.05% for 3 minutes at 37°C. Finally, cells were collected by centrifugation at 200 x g for 5 min for further experiments.
Cell cycle analysis
Cells were plated on 60 mm plates in triplicate and were cultured for 24, 48 or 72 h. The cells were treated with –EDTA 0.05% for 3 minutes at 37°C and collected in 5 ml Falcon round-bottom polystyrene test tubes. Then cells were washed 1x PBS by centrifugation at 200 x g for 5 min. The cell pellet was fixed by adding a few drops of cold 70% ethanol and stored at -20°C. On the day of measurement 2 ml of PBS was added to each cell pellet, then the suspension was centrifuged at 1000 x g for 5 min. The cell pellet was washed with 2 ml PBS again. Next the pellet was suspended in 250 µl of 10 µg/ml RNase A in PBS (stock 10 mg/ml) and incubated for 45 min at room temperature. Then 8 µl of propidium iodide (1 mg/ml) was added to the cell suspension (250 µl) and incubated for 15–30 min (protected from the light). Before the measurement samples were kept on ice. Measurement and analyses were performed on a NovoCyte Flow Cytometer (Agilent Technologies, Santa Clara, California, USA). A file with plots of FSC/SSC, PI-A/width, PI-A/events was prepared.
FLIM analysis of living cells
FLIM was used to measure the fluorescence lifetime of the membrane-order sensitive probe, di-4 ANEPPDHQ (di-4) according to Owen et al. [40]. HeLa cells were grown in LabTek chambers in DMEM medium with 10% FBS and 2 mM glutamine. After 24 h cells were washed twice with HBSS in 10 mM HEPES, pH 7.4 and stained with 2 µM di-4 in HBSS 10 and mM HEPES, pH 7.4, for 5 min (the stock was 2 mM di4 in DMSO). Cells were washed twice and the measurements were performed in HBSS in 10 mM HEPES, pH 7.4. Di-4 fluorescence lifetime microscopic images were obtained by time-correlated single-photon counting (TCSPC) using an LSM 510 META microscope (Carl Zeiss, Jena, Germany) equipped with a PicoQuant FLIM/FCS module (Berlin, Germany). Samples were excited at 470 nm and imaged with a 40× WI objective (NA 1.2) using an LP 510 filter set. Acquisition time was adjusted to collect at least 1000 photons per pixel. Each pixel in the image was pseudocolored according to the average fluorescent lifetime. The images were analyzed in the PicoQuant Analysis program.
GPMV isolation, protein profile and FLIM analysis
GPMVs were isolated from “scrambled” and KnD EFR3A HeLa cells, using vesiculation buffer (10 mM HEPES, 150 mM NaCl pH 7.25 and freshly added 2 mM CaCl2, 2 mM NEM (N-ethylmaleimide)) for 2h at 37°C according to the protocol [41]. GPMVs were stained with di-4 (2 µM) (10 min/RT) and placed in a covered 0.01% Poly-L-lysine, sealed chamber. FLIM was performed as described above.
svFCS measurements
Twenty-four hours before svFCS measurements, subconfluent cultures of HeLa cells were trypsinized and plated onto Lab-Tek (35x103). On the day of the experiment, cells were washed 3 times with HBSS in 10 mM HEPES, pH 7.4 followed by 10 min incubation at room temperature in the dark, in 0.075 µM BSA, 0.075 µM Bodipy SM (Thermo Fisher Scientific, Waltham, MA, USA) in HBSS in 10 mM HEPES. Next, cells were washed four times with HBSS in 10 mM HEPES, pH 7.4. Measurements were carried out using a custom-made svFCS system based on the Axiovert 200 M fluorescence microscope (Carl Zeiss, Oberkochen, Germany), according to the procedure described previously [42]. Briefly, the 488 nm laser beam power was adjusted to 330 µW and the waist size was calibrated using 2 nM rhodamine 6G solution. For the living cell analysis, all the measurements were performed under physiological conditions at 37°C. The signal was recorded at an intensity of 2–4 µW and the data were collected in a series of 20 runs lasting 5 s each. The measurements were carried out on at least 10 individual cells per spot size. Next, the generated autocorrelation functions (ACFs) were examined and analyzed by the IGOR Pro program. The data were fitted to a 2D lateral diffusion model and the average time τd (diffusion time) was calculated. A single diffusion law was constructed from the measurements obtained at four different waist sizes.
Rescue mutant
The shRNA resistant control (rescue mutant EFR3A) was obtained through generation of silent mutations within the coding sequence of the EFR3A protein, resulting in a synonymous amino acid product, but the protein mRNA no longer being a target for the shRNA. Such a prepared gene sequence was synthesized via Integrated DNA Technologies ITD (Redwood City, California 94065 USA). The vector obtained from the IDT sequence in pUCIDT (Amp) was subcloned into the p3XFLAG-CMV10 (Sigma) vector using NotI and KpnI restriction enzymes using the Quick Ligation Kit (New England BioLabs). For svFCS control, EFR3A KnD cells (1.5 × 106) were transfected with 1 µg of “empty” p3XEFLAG-CMV10 vector for 48 h at 37°C in a humidified atmosphere of 5% CO2. For EFR3A “rescue” expression, EFR3A KnD cells (1.5 × 106) were transfected with 1 µg of EFR3A “rescue” plasmids p3XEFLAG-CMV10 EFR3A for 48 h at 37°C in a humidified atmosphere of 5% CO2.
Wound healing assay
HeLa “scrambled” and EFR3A KnD cells were plated on 12-well plates and grown to 75–80% confluence in a complete medium and then they were serum-starved for 24 h. The cell-free gaps were created by an ibidi Culture-Insert 3 Well (80369). The cells were then treated with 50 ng/ml of EGF in a serum-free medium. The images (2464 × 2056 pixels) were captured at 24 and 48 h at 20× magnification using a Zeiss digital camera integrated with an Axio Vert.A1 inverted microscope (Carl Zeiss). The area of the wound was quantified using ImageJ software. The cell migration was expressed as the ratio of wound closure (R): R = ((A 0 h − A ∆ h)/ A 0 h), where A 0 h is the area of the cell-free gap measured immediately after the insert was removed and A ∆ h is the area of the artificial wound measured after 24 or 48 h.
Cytosolic Ca2+ concentrations
Forty-eight hours before the experiment, subconfluent HeLa KnD EFR3A and HeLa “scrambled” cells were trypsinized and plated into Lab-Tek chambers. A day before the experiment, the media were replaced with serum-free media and cultures were incubated overnight (16–20 h). On the day of the experiment, cells were washed with HBSS in 10 mM HEPES pH 7.4 and then the Fluo-4 Direct Calcium Assay Kit was used according to the manufacturer’s protocol. Finally, cells were washed twice in 1x HBSS in 10 mM HEPES, pH 7.4. First, cells were analyzed without EGF. Next EGF was added to the same wells to the final concentration of 50 ng/ml.
Fluorescence imaging was performed using a STELLARIS 8 system with a thermostated chamber at 37°C, excitation and emission filters at 488 and 515 nm, respectively. Objective HC PL APO 86x/1.20 WATER, numerical aperture 1.2 was used. Images were acquired every 1.3 s for 5 min. All image processing was performed using ImageJ Software (NIH). Fluorescence intensity of individual cells was obtained by defining a region of interest for each individual cell. The linear intensities were acquired from the 'plot profile' over the entire 200 frames of the video.
Western blotting and overlay assay
Protein samples were separated by SDS-PAGE and electrotransferred to 0.2 µm nitrocellulose membranes. Membranes were blocked with 5% nonfat milk in TBS-T, followed by overnight incubation at 4°C with an appropriate primary antibody diluted as indicated in Table 1 in TBS-T. After washing three times with 0.1% Tween-20 in TBS (TBS-T), membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody, developed with chemiluminescence Radiance ECL kit and visualized using an Azure 600 detector (Azure Biosystems, Dublin, CA, USA).
For overlay assay purified recombinant EFR3A was subjected to SDS-PAGE and were transferred onto nitrocellulose membrane as was mentioned above and stained with Ponceau S. Next, membrane strips containing bands corresponding to molecular weight > 70 kDa were blocked with 5% dry milk in TBS-T overnight at 4°C followed by incubation with increasing concentrations of recombinant flotillin-2 (flotillin 2 in TBS-T buffer), after which they were incubated with goat anti-flotillin-2 antibodies 1:1000. Finally the blots were treated with HRP-conjugated anti-goat antibodies and chemiluminescent reaction was developed and recorded as above..
Cholesterol and protein determination
Analysis of the amount of cholesterol was performed using the Amplex Red Cholesterol Assay Kit. The protein concentration was determined using the BCA Protein Assay Kit. Subsequently proteins in each collected fraction from the sucrose gradient were precipitated with 10% TCA prior to SDS-PAGE.
Statistical analysis
The statistics were performed by ANOVA and Student’s t test using the GraphPad Prism program.