EFR3A: a new raft domain organizing protein?

Fig. 1. Flotillin-2 binds EFR3A in DRMs of HeLa cells. DRM fraction from HeLa cells diluted 1:1 with 2% octyl glucoside in 40 mM Tris–HCl, pH 7.4, 300 mM NaCl and protease inhibitor cocktail (Sigma–Aldrich) were incubated overnight with flotillin-2 Sepharose resin at 4 °C. Then the resin was thoroughly washed with the same buffer and suspended in sample buffer (20% SDS, 50% glycerol, 25 mM EDTA, 250 mM DTT, and 0.25 M Tris pH 6.8), boiled for 5 min, and subjected to SDS-PAGE. A Example of sequence coverage for EFR3A resulting from MS/MS identification of proteins bound to flotillin-2 Sepharose. SDS-PAGE gel fragment containing protein bands larger than 60 kDa was cut out and subjected to MS/MS identification (see also Additional file 1: Figure S1 and Table S1). B Western blotting of samples derived from a pull-down assay. Only bound fraction and unconjugated resin control are shown. Nitrocellulose was probed with an EFR3A antibody (Abnova). C Co-IP results, whole cell lysates of HeLa cells were incubated with 5 μg of anti-FL2 goat antibodies (Abcam) to protein G Dynabeads (Life Technologies) overnight at 4 °C with rotation, according to the manufacturer’s protocol. Nonimmune rabbit IgG…

Fig. 2. Bacterially expressed recombinant EFR3A binds to recombinant flotillin-2 in vitro, in the overlay assay. Recombinant EFR3A protein was purified on metal ion chelating resin (Ni2+) and subjected to SDS-PAGE followed by transfer onto nitrocellulose. Then, membrane strips containing bands corresponding to molecular weight larger than 70 kDa were incubated with increasing concentrations of recombinant flotillin-2, followed by incubation with goat anti-flotillin-2 antibodies (Abcam) and secondary donkey anti-goat antibodies (Santa Cruz)

Fig. 3. EFR3A is present in the DRM fraction and its localization in the DRM fraction is sensitive to the removal of cholesterol from the HeLa cell plasma membrane. A Western blotting of sucrose step gradient fractions obtained after ultracentrifugation of cold 1% Triton X-100 extract (30 min on ice) from HeLa cells. 25 × 106 cells were used. Then, fractions were collected and proteins were precipitated with 10% TCA and analyzed with appropriate primary and secondary antibodies. B Western blotting of the Triton extract from cells treated with 8 mM β-methyl-cyclodextrin shows that localization of EFR3A in the DRM fraction is sensitive to the removal of cholesterol; for comparison, anti-EGFR, and anti-flotillin antibodies reactions are shown. For total protein and cholesterol profiles see Additional file 1: Fig. S3

Fig. 4. Silencing expression of the EFR3A gene in the Hela cell line induces changes in lateral membrane organization. Stable cell lines were obtained using EFR3A shRNA lentiviral particles or “scrambled” shRNA lentiviral particles as described in the Methods section. A Cell extracts of control or transduced cell lines were submitted to SDS-PAGE and western blotting probed with anti-EFR3A antibodies. GAPDH visualization was used as a loading control. B Quantitation of the EFR3A fractions shown in A. C Marked decrease of EFR3A level in cells is accompanied by a substantial reduction of EGFR in the DRM fraction. D Examples of FLIM images of the cell plasma membrane and E GPMVs derived from EFR3A KnD and “scrambled” cells. F and G Quantitation of FLIM data of EFR3A KnD and “scrambled” cells and corresponding GPMVs. For FLIM measurements of the fluorescence lifetime of the membrane-order sensitive probe, di-4 ANEPPDHQ (Invitrogen) control “scrambled” and EFR3A KnD HeLa cells were grown in LabTek chambers in DMEM medium with 10% FBS. After 24 h cells were washed twice and stained with a 2 μM di-4 probe in DMEM medium for 5 min. Cells were washed twice in HBSS buffer with 10 mM HEPES pH 7.4…

Fig. 5. “Rescue expression” of EFR3A in KnD HeLa cells. A Western blotting of total protein extracts of EFR3A KnD HeLa cells transfected with p3xEFLAG-CMV10 EFR3A (lane 1) and control p3xEFLAG-CMV10 plasmids (lane 2). Anti-FLAG antibodies (Merck) were used as primary antibodies. B Representative di-4 FLIM images of control, “scrambled” cell (left), EFR3A KnD cell transfected with an empty vector (middle), and “rescue transfected” (right). C Examples of di4 FLIM images of GPMVs generated from control, “scrambled” cells (left), EFR3A KnD cells transfected with an “empty” vector (middle), and EFR3A KnD cells transfected with a “rescue” plasmid p3xEFLAG-CMV10 EFR3A. D and E Quantitative distribution of fluorescence lifetime values obtained from FLIM analyses of di-4 labeled cells. D Plasma membrane and GPMVs. E FLIM analyses presented here were performed 48 h following transfection. F Dot-blot analysis of GPMVs derived from “rescue”-transfected EFR3A KnD cells using an anti-FLAG antibody. GPMVs were induced using a buffer containing NEM and CaCl2 48 h after transfection. Then, from the collected vesicles, proteins were precipitated with 10% TCA, washed, and resuspended in a reducing buffer…

Fig. 6. Mobility of the raft probe in EFR3A KnD partially returns towards normal in “rescue”-transfected EFR3A KnD HeLa cells svFCS measurements. A Diffusion time as a function of waist surface area. Black line: control HeLa “scrambled”, red line: EFR3A KnD, and green line: “rescue”-transfected EFR3A KnD HeLa cells. Cells were grown in LabTek chambers in DMEM medium with 10% FBS. After 24 h cells were washed three times and stained with 0.075 μM BODIPY-SM in HBSS with 10 mM HEPES and 0.075 μM BSA medium for 10 min. Cells were washed three times and measurements were performed in HBSS buffer with 10 mM HEPES, pH 7.4. For comparison, wild-type cells were treated with 8 mM β-methyl cyclodextrin for 1 h at 37 °C B. T0 and apparent diffusion coefficient. SEM, standard error of the mean

Fig. 7. Silencing of EFR3A gene expression affects G1 and G2/M length and proliferation but not cell mobility. A Cell cycle phase distribution in HeLa cells was evaluated via PI staining followed by flow cytometry presented as histograms. B Quantitation of flow cytometry analysis. Error bars represent the SEM of three independent experiments. *p < 0.05. The percentage of G1 phase cells increased within 48 h to 56%. C Cell proliferation was evaluated by WST-1 staining according to the Methods section. **Indicates a p ≤ 0.01 compared with control cells. D Representative images from the wound healing assay showing changes in HeLa cells with decreased EFR3A gene expression in comparison with control “scrambled” cells under stimulation with EGFR (50 ng/ml) for 48 h. E Quantitative analysis from the wound healing assay in the form of a bar graph showing the ratio of wound closure in EFR3A KnD versus “scrambled” cells. The observed reduced motility in cells with decreased expression of EFR3A in comparison with “scrambled” cells was found to be statistically insignificant (p-value = 0.158)

Fig. 8. Silencing of EFR3 gene expression affects PLCγ1 and EGFR phosphorylation. A Effect of EFR3A gene silencing on activation of EGF-induced EGF receptor and phospholipase PLCγ1 in HeLa cells via western blotting analysis. B Effect of MβCD on EGFR and PLCγ1 phosphorylation upon EGF treatment of WT HeLa cells. Fifteen micrograms of cell lysates was loaded on the 10% SDS-PAGE gel. C and D Quantitative analysis of data presented in A as bar graphs of fold change calculated as the ratio of relative levels of phospho-PLCγ and pEGFR normalized to the corresponding PLCγ1 and EGFR signal respectively. Significance values were calculated by paired Student’s t-test for **p < 0.01, ***p < 0.001 and ****p < 0.0001

Fig. 9. Silencing of EFR3 gene expression affects EGF-induced Ca2+ influx. Fluo-4 Direct calcium assay kit was used according to the manufacturer’s protocol. First, cells were analyzed without EGF. Next, EGF was added to the same wells to the final concentration of 50 ng/ml. Fluorescence was acquired using a STELLARIS 8 system with a thermostated chamber at 37 °C and at excitation of 488 nm and emission within the range of 492–577 nm. 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). The 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. For further details see Methods and Additional file 2: Movie 1, Additional file 3: Movie 2

Fig. 10. Proposed model of EFR3A participation of raft domain regulation. Flotillin (most likely flotillin-1/-2 heterodimer) is a member of preexisting unstable protein–lipid complexes (τ1/2< 0.1 ms; < 10 nm in diameter) composed of few proteins and lipids which upon interaction with a protein–raft domain organizer, such as EFR3A, cluster into raft domains [resting state rafts, i.e., domains ~ 20 nm in diameter, which are considered more stable (τ1/2 ~ 1 s) and functional]. Protein clustering modulates membrane bilayer physical properties, such as fluidity and local diffusion